Antibody drug conjugates (adc) that bind to 158p1d7 proteins

ABSTRACT

Antibody drug conjugates (ADC&#39;s) that bind to 158P1D7 protein and variants thereof are described herein. 158P1D7 exhibits tissue specific expression in normal adult tissue, and is aberrantly expressed in glioblastoma, lung cancer, bladder cancer, and breast cancer. Consequently, the ADC&#39;s of the invention provide a therapeutic composition for the treatment of cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/634,606, filed 27 Feb. 2015, now pending, which is a continuation ofU.S. application Ser. No. 13/975,214, filed 23 Aug. 2013, now U.S. Pat.No. 8,968,742, which claims priority to U.S. Provisional PatentApplication No. 61/692,448, filed 23 Aug. 2012. All applications areherein incorporated by reference in their entirety for all purposes.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 511582505002SeqList.txt,date recorded: Mar. 25, 2016, size: 40,227 bytes.

FIELD OF THE INVENTION

The invention described herein relates to antibodies, binding fragments,and antibody drug conjugates (ADCs) thereof, that bind proteins, termed158P1D7. The invention further relates to prognostic, prophylactic andtherapeutic methods and compositions useful in the treatment of cancersthat express 158P1D7.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of human death next to coronarydisease. Worldwide, millions of people die from cancer every year. Inthe United States alone, as reported by the American Cancer Society,cancer causes the death of well over a half-million people annually,with over 1.2 million new cases diagnosed per year. While deaths fromheart disease have been declining significantly, those resulting fromcancer generally are on the rise. In the early part of the next century,cancer is predicted to become the leading cause of death.

Worldwide, several cancers stand out as the leading killers. Inparticular, carcinomas of the lung, prostate, breast, colon, pancreas,ovary, and bladder represent the primary causes of cancer death. Theseand virtually all other carcinomas share a common lethal feature. Withvery few exceptions, metastatic disease from a carcinoma is fatal.Moreover, even for those cancer patients who initially survive theirprimary cancers, common experience has shown that their lives aredramatically altered. Many cancer patients experience strong anxietiesdriven by the awareness of the potential for recurrence or treatmentfailure. Many cancer patients experience physical debilitationsfollowing treatment. Furthermore, many cancer patients experience arecurrence.

Worldwide, prostate cancer is the fourth most prevalent cancer in men.In North America and Northern Europe, it is by far the most commoncancer in males and is the second leading cause of cancer death in men.In the United States alone, well over 30,000 men die annually of thisdisease—second only to lung cancer. Despite the magnitude of thesefigures, there is still no effective treatment for metastatic prostatecancer. Surgical prostatectomy, radiation therapy, hormone ablationtherapy, surgical castration and chemotherapy continue to be the maintreatment modalities. Unfortunately, these treatments are ineffectivefor many and are often associated with undesirable consequences.

On the diagnostic front, the lack of a prostate tumor marker that canaccurately detect early-stage, localized tumors remains a significantlimitation in the diagnosis and management of this disease. Although theserum prostate specific antigen (PSA) assay has been a very useful tool,its specificity and general utility is widely regarded as lacking inseveral important respects.

Progress in identifying additional specific markers for prostate cancerhas been improved by the generation of prostate cancer xenografts thatcan recapitulate different stages of the disease in mice. The LAPC (LosAngeles Prostate Cancer) xenografts are prostate cancer xenografts thathave survived passage in severe combined immune deficient (SCID) miceand have exhibited the capacity to mimic the transition from androgendependence to androgen independence (Klein et al., 1997, Nat. Med.3:402). More recently identified prostate cancer markers include PCTA-1(Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252),prostate-specific membrane antigen (PSMA) (Pinto et al., Clin Cancer Res1996 Sep. 2 (9): 1445-51), STEAP (Hubert, et al., Proc Natl Acad SciUSA. 1999 Dec. 7; 96(25): 14523-8) and prostate stem cell antigen (PSCA)(Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95: 1735).

While previously identified markers such as PSA have facilitated effortsto diagnose and treat prostate cancer, there is need for theidentification of additional markers and therapeutic targets forprostate and related cancers in order to further improve diagnosis andtherapy. An estimated 130,200 cases of colorectal cancer occurred in2000 in the United States, including 93,800 cases of colon cancer and36,400 of rectal cancer.

Colorectal cancers are the third most common cancers in men and women.Incidence rates declined significantly during 1992-1996 (−2.1% peryear). Research suggests that these declines have been due to increasedscreening and polyp removal, preventing progression of polyps toinvasive cancers. There were an estimated 56,300 deaths (47,700 fromcolon cancer, 8,600 from rectal cancer) in 2000, accounting for about11% of all U.S. cancer deaths.

At present, surgery is the most common form of therapy for colorectalcancer, and for cancers that have not spread, it is frequently curative.Chemotherapy, or chemotherapy plus radiation, is given before or aftersurgery to most patients whose cancer has deeply perforated the bowelwall or has spread to the lymph nodes. A permanent colostomy (creationof an abdominal opening for elimination of body wastes) is occasionallyneeded for colon cancer and is infrequently required for rectal cancer.There continues to be a need for effective diagnostic and treatmentmodalities for colorectal cancer.

Of all new cases of cancer in the United States, bladder cancerrepresents approximately 5 percent in men (fifth most common neoplasm)and 3 percent in women (eighth most common neoplasm). The incidence isincreasing slowly, concurrent with an increasing older population. In1998, there were an estimated 54,500 cases, including 39,500 in men and15,000 in women. The age-adjusted incidence in the United States is 32per 100,000 for men and eight per 100,000 in women. The historicmale/female ratio of 3:1 may be decreasing related to smoking patternsin women. There were an estimated 11,000 deaths from bladder cancer in1998 (7,800 in men and 3,900 in women). Bladder cancer incidence andmortality strongly increase with age and will be an increasing problemas the population becomes more elderly.

Most bladder cancers recur in the bladder. Bladder cancer is managedwith a combination of transurethral resection of the bladder (TUR) andintravesical chemotherapy or immunotherapy. The multifocal and recurrentnature of bladder cancer points out the limitations of TUR. Mostmuscle-invasive cancers are not cured by TUR alone. Radical cystectomyand urinary diversion is the most effective means to eliminate thecancer but carry an undeniable impact on urinary and sexual function.There continues to be a significant need for treatment modalities thatare beneficial for bladder cancer patients.

There were an estimated 164,100 new cases of lung and bronchial cancerin 2000, accounting for 14% of all U.S. cancer diagnoses. The incidencerate of lung and bronchial cancer is declining significantly in men,from a high of 86.5 per 100,000 in 1984 to 70.0 in 1996. In the 1990s,the rate of increase among women began to slow. In 1996, the incidencerate in women was 42.3 per 100,000.

Lung and bronchial cancer caused an estimated 156,900 deaths in 2000,accounting for 28% of all cancer deaths. During 1992-1996, mortalityfrom lung cancer declined significantly among men (−1.7% per year) whilerates for women were still significantly increasing (0.9% per year).Since 1987, more women have died each year of lung cancer than breastcancer, which, for over 40 years, was the major cause of cancer death inwomen. Decreasing lung cancer incidence and mortality rates most likelyresulted from decreased smoking rates over the previous 30 years;however, decreasing smoking patterns among women lag behind those ofmen. Of concern, although the declines in adult tobacco use have slowed,tobacco use in youth is increasing again.

Treatment options for lung and bronchial cancer are determined by thetype and stage of the cancer and include surgery, radiation therapy, andchemotherapy. For many localized cancers, surgery is usually thetreatment of choice. Because the disease has usually spread by the timeit is discovered, radiation therapy and chemotherapy are often needed incombination with surgery. Chemotherapy alone or combined with radiationis the treatment of choice for small cell lung cancer; on this regimen,a large percentage of patients experience remission, which in some casesis long lasting. There is however, an ongoing need for effectivetreatment and diagnostic approaches for lung and bronchial cancers.

An estimated 182,800 new invasive cases of breast cancer were expectedto occur among women in the United States during 2000. Additionally,about 1,400 new cases of breast cancer were expected to be diagnosed inmen in 2000. After increasing about 4% per year in the 1980s, breastcancer incidence rates in women have leveled off in the 1990s to about110.6 cases per 100,000.

In the U.S. alone, there were an estimated 41,200 deaths (40,800 women,400 men) in 2000 due to breast cancer. Breast cancer ranks second amongcancer deaths in women. According to the most recent data, mortalityrates declined significantly during 1992-1996 with the largest decreasesin younger women, both white and black. These decreases were probablythe result of earlier detection and improved treatment.

Taking into account the medical circumstances and the patient'spreferences, treatment of breast cancer may involve lumpectomy (localremoval of the tumor) and removal of the lymph nodes under the arm;mastectomy (surgical removal of the breast) and removal of the lymphnodes under the arm; radiation therapy; chemotherapy; or hormonetherapy. Often, two or more methods are used in combination. Numerousstudies have shown that, for early stage disease, long-term survivalrates after lumpectomy plus radiotherapy are similar to survival ratesafter modified radical mastectomy. Significant advances inreconstruction techniques provide several options for breastreconstruction after mastectomy. Recently, such reconstruction has beendone at the same time as the mastectomy.

Local excision of ductal carcinoma in situ (DCIS) with adequate amountsof surrounding normal breast tissue may prevent the local recurrence ofthe DCIS. Radiation to the breast and/or tamoxifen may reduce the chanceof DCIS occurring in the remaining breast tissue. This is importantbecause DCIS, if left untreated, may develop into invasive breastcancer. Nevertheless, there are serious side effects or sequelae tothese treatments. There is, therefore, a need for efficacious breastcancer treatments.

There were an estimated 23,100 new cases of ovarian cancer in the UnitedStates in 2000. It accounts for 4% of all cancers among women and rankssecond among gynecologic cancers. During 1992-1996, ovarian cancerincidence rates were significantly declining. Consequent to ovariancancer, there were an estimated 14,000 deaths in 2000. Ovarian cancercauses more deaths than any other cancer of the female reproductivesystem.

Surgery, radiation therapy, and chemotherapy are treatment options forovarian cancer. Surgery usually includes the removal of one or bothovaries, the fallopian tubes (salpingo-oophorectomy), and the uterus(hysterectomy). In some very early tumors, only the involved ovary willbe removed, especially in young women who wish to have children. Inadvanced disease, an attempt is made to remove all intra-abdominaldisease to enhance the effect of chemotherapy. There continues to be animportant need for effective treatment options for ovarian cancer.

There were an estimated 28,300 new cases of pancreatic cancer in theUnited States in 2000. Over the past 20 years, rates of pancreaticcancer have declined in men. Rates among women have remainedapproximately constant but may be beginning to decline. Pancreaticcancer caused an estimated 28,200 deaths in 2000 in the United States.Over the past 20 years, there has been a slight but significant decreasein mortality rates among men (about −0.9% per year) while rates haveincreased slightly among women.

Surgery, radiation therapy, and chemotherapy are treatment options forpancreatic cancer. These treatment options can extend survival and/orrelieve symptoms in many patients but are not likely to produce a curefor most. There is a significant need for additional therapeutic anddiagnostic options for cancers. These include the use of antibodies,vaccines, and small molecules as treatment modalities. Additionally,there is also a need to use these modalities as research tools todiagnose, detect, monitor, and further the state of the art in all areasof cancer treatment and studies.

The therapeutic utility of monoclonal antibodies (mAbs) (G. Kohler andC. Milstein, Nature 256:495-497 (1975)) is being realized. Monoclonalantibodies have now been approved as therapies in transplantation,cancer, infectious disease, cardiovascular disease and inflammation.Different isotypes have different effector functions. Such differencesin function are reflected in distinct 3-dimensional structures for thevarious immunoglobulin isotypes (P. M. Alzari et al., Annual Rev.Immunol., 6:555-580 (1988)).

Because mice are convenient for immunization and recognize most humanantigens as foreign, mAbs against human targets with therapeuticpotential have typically been of murine origin. However, murine mAbshave inherent disadvantages as human therapeutics. They require morefrequent dosing as mAbs have a shorter circulating half-life in humansthan human antibodies. More critically, the repeated administration ofmurine antibodies to the human immune system causes the human immunesystem to respond by recognizing the mouse protein as a foreign andgenerating a human anti-mouse antibody (HAMA) response. Such a HAMAresponse may result in allergic reaction and the rapid clearing of themurine antibody from the system thereby rendering the treatment bymurine antibody useless. To avoid such affects, attempts to create humanimmune systems within mice have been attempted.

Initial attempts hoped to create transgenic mice capable of respondingto antigens with antibodies having human sequences (See Bruggemann etal, Proc. Nat'l. Acad. Sci. USA 86:6709-6713 (1989)), but were limitedby the amount of DNA that could be stably maintained by availablecloning vehicles. The use of yeast artificial chromosome (YAC) cloningvectors led the way to introducing large germline fragments of human Iglocus into transgenic mammals. Essentially a majority of the human V, D,and J region genes arranged with the same spacing found in the humangenome and the human constant regions were introduced into mice usingYACs. One such transgenic mouse strain is known as XenoMouse® mice andis commercially available from Amgen Fremont, Inc. (Fremont CA).

SUMMARY OF THE INVENTION

The invention provides antibodies, binding fragments, and antibody drugconjugates (ADCs) thereof that bind to 158P1D7 proteins and polypeptidefragments of 158P1D7 proteins. In some embodiments, the inventioncomprises fully human antibodies conjugated with a therapeutic agent. Incertain embodiments, there is a proviso that the entire nucleic acidsequence of FIG. 3 is not encoded and/or the entire amino acid sequenceof FIG. 2 is not prepared. In certain embodiments, the entire nucleicacid sequence of FIG. 3 is encoded and/or the entire amino acid sequenceof FIG. 2 is prepared, either of which are in respective human unit doseforms.

The invention further provides various immunogenic or therapeuticcompositions, such as antibody drug conjugates, and strategies fortreating cancers that express 158P1D7 such as cancers of tissues listedin Table I, especially bladder cancer.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 . The cDNA and amino acid sequence of 158P1D7 is shown in FIG. 1. The start methionine is underlined. The open reading frame extendsfrom nucleic acid 23-2548 including the stop codon. The nucleic acid andamino acid sequences begin on the first panel of FIG. 1 , and continueon FIG. 1-2 and FIG. 1-3 . FIG. 1-2 shows nucleic acids 961-2040 andamino acids 314-693. FIG. 1-3 shows nucleic acids 2041-2555 and aminoacids 694-841.

FIG. 2 . Nucleic Acid and Amino Acid sequences of 158P1D7 antibodies.FIG. 2A shows the cDNA and amino acid sequence of Ha15-10ac12 heavychain. Double-underlined is the heavy chain variable region, underlinedis the heavy chain human IgG2 constant region. FIG. 2B shows the cDNAand amino acid sequence of Ha15-10ac12 light chain. Double-underlined isthe light chain variable region, underlined is the human kappa constantregion.

FIG. 3 . Amino Acid sequences of 158P1D7 antibodies. FIG. 3A shows theamino acid sequence of Ha15-10ac12 heavy chain. Double-underlined is theheavy chain variable region, and underlined is the human IgG2 constantregion. FIG. 3B the amino acid sequence of Ha15-10ac12 light chain.Double-underlined is the light chain variable region, and underlined isthe human kappa constant region.

FIG. 4 . Alignment of Ha15-10ac12 antibodies to human Ig germline. FIG.4A and FIG. 4A-2 alignment of Ha15-10ac12 heavy chain (SEQ ID NO:3,positions 1-360; SEQ ID NO:4, positions 1-120) to human Ig germline.FIG. 4B alignment of Ha15-10ac12 light chain (SEQ ID NO:5, positions1-240; SEQ ID NO:6, positions 1-80) to human Ig germline IGKV2D-28*01(SEQ ID NO:10).

FIG. 5 . Efficacy of Ha15-10ac12vcMMAE in a Subcutaneously EstablishedXenograft Model of Human Bladder Cancer AG-B7 in SCID Mice.

FIG. 6 . Efficacy of Ha15-10ac12vcMMAE in Subcutaneously EstablishedHuman Bladder Cancer RT-4-XCL Implanted in SCID Mice.

FIG. 7 . Efficacy of Ha15-10ac12vcMMAE in Subcutaneously EstablishedHuman Lung Cancer NCI-H322M-XCL Implanted in SCID Mice.

FIG. 8 . Efficacy of Ha15-10ac12vcMMAE in a Subcutaneously EstablishedXenograft Model of Human Bladder Cancer AG-B7 in SCID Mice.

FIG. 9 . Detection of 158P1D7 protein in cancer patient specimens byIHC. FIG. 9A and FIG. 9B show bladder cancer specimens. FIG. 9C and FIG.9D show breast cancer specimens. FIG. 9E and FIG. 9F show lung cancerspecimens. FIG. 9G and FIG. 9H show glioblastoma cancer specimens.

FIG. 10 . Efficacy of Ha15-10ac12vcMMAE in subcutaneous establishedxenograft model of human bladder cancer SW780 in SCID mice.

FIG. 11 . In vitro efficacy of Ha15-10ac12vcMMAE in CHP-212 cytotoxicityassay compared with M15-68(2) 18 (a.k.a. 68(18) 1.1) Mab.

FIG. 12 . Efficacy of Ha15-10ac12vcMMAE in Subcutaneous establishedXenograft model of Patient Derived human bladder cancer AG-B8 in SCIDmice

FIG. 13 . A saturation curve for Ha15-10ac12vcMMAE.

FIG. 14 . A histogram showing Mean Florescence Intensity (MFI) ofHa15-10ac12vcMMAE.

FIG. 15A and FIG. 15B. Evaluation of the In-vitro cytotoxicity ofHa15-10ac12vcMMAE on CHP-212 cells (FIG. 15A) and IGROV-1 cells (FIG.15B).

DETAILED DESCRIPTION OF THE INVENTION Outline of Sections

-   -   I.) Definitions    -   II.) 158P1D7 Antibodies    -   III.) Antibody Drug Conjugates Generally        -   III(A). Maytansinoids        -   III(B). Auristatins and dolostatins        -   III(C). Calicheamicin        -   III(D). Other Cytotoxic Agents    -   IV.) Antibody Drug Conjugates which Bind 158P1D7    -   V.) Linker Units    -   VI.) The Stretcher Unit    -   VII.) The Amino Acid Unit    -   VIII.) The Spacer Unit    -   IX.) The Drug Unit    -   X.) Drug Loading    -   XI.) Methods of Determining Cytotoxic effect of ADCs    -   XII.) Treatment of Cancer(s) Expressing 158P1D7    -   XIII.) 158P1D7 as a Target for Antibody-based Therapy    -   XIV.) 158P1D7 ADC Cocktails    -   XV.) Combination Therapy    -   XVI.) Kits/Articles of Manufacture

I.) Definitions

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. Many of the techniques and procedures describedor referenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art, such as, forexample, the widely utilized molecular cloning methodologies describedin Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. edition(1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Asappropriate, procedures involving the use of commercially available kitsand reagents are generally carried out in accordance with manufacturerdefined protocols and/or parameters unless otherwise noted.

When a trade name is used herein, reference to the trade name alsorefers to the product formulation, the generic drug, and the activepharmaceutical ingredient(s) of the trade name product, unless otherwiseindicated by context.

The terms “advanced cancer”, “locally advanced cancer”, “advanceddisease” and “locally advanced disease” mean cancers that have extendedthrough the relevant tissue capsule, and are meant to include stage Cdisease under the American Urological Association (AUA) system, stageC1-C2 disease under the Whitmore-Jewett system, and stage T3-T4 and N+disease under the TNM (tumor, node, metastasis) system. In general,surgery is not recommended for patients with locally advanced disease,and these patients have substantially less favorable outcomes comparedto patients having clinically localized (organ-confined) cancer.

The abbreviation “AFP” refers todimethylvaline-valine-dolaisoleuine−dolaproine-phenylalanine-p-phenylenediamine(see Formula XVI infra).

The abbreviation “MMAE” refers to monomethyl auristatin E (see FormulaXI infra).

The abbreviation “AEB” refers to an ester produced by reactingauristatin E with paraacetyl benzoic acid (see Formula XX infra).

The abbreviation “AEVB” refers to an ester produced by reactingauristatin E with benzoylvaleric acid (see Formula XXI infra).

The abbreviation “MMAF” refers todovaline-valine-dolaisoleuine-dolaproine-phenylalanine (see Formula XVIVinfra).

Unless otherwise noted, the term “alkyl” refers to a saturated straightor branched hydrocarbon having from about 1 to about 20 carbon atoms(and all combinations and subcombinations of ranges and specific numbersof carbon atoms therein), with from about 1 to about 8 carbon atomsbeing preferred. Examples of alkyl groups are methyl, ethyl, n-propyl,iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl,2-pentyl, 3-pentyl, 2-methyl-2-butyl, n-hexyl, n-heptyl, n-octyl,n-nonyl, n-decyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl,1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl,4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl,2,3-dimethyl-2-butyl, and 3,3-dimethyl-2-butyl.

Alkyl groups, whether alone or as part of another group, can beoptionally substituted with one or more groups, preferably 1 to 3 groups(and any additional substituents selected from halogen), including, butnot limited to, -halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl),—O—(C₂-C₈ alkynyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′, —S(O)₂R′, —S(O)R′, —OH,═O, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN, where each R′ is independentlyselected from —H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or-aryl, and wherein said —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈alkynyl), -aryl, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, and —C₂-C₈ alkynyl groupscan be optionally further substituted with one or more groups including,but not limited to, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl,-halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl),-aryl, —C(O)R″, —OC(O)R″, —C(O)OR″, —C(O)NH₂, —C(O)NHR″, —C(O)N(R″)₂,—NHC(O)R″, —SR″, —SO₃R″, —S(O)₂R″, —S(O)R″, —OH, —N₃, —NH₂, —NH(R″),—N(R″)₂ and —CN, where each R″ is independently selected from —H, —C₁-C₈alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or -aryl.

Unless otherwise noted, the terms “alkenyl” and “alkynyl” refer tostraight and branched carbon chains having from about 2 to about 20carbon atoms (and all combinations and subcombinations of ranges andspecific numbers of carbon atoms therein), with from about 2 to about 8carbon atoms being preferred. An alkenyl chain has at least one doublebond in the chain and an alkynyl chain has at least one triple bond inthe chain. Examples of alkenyl groups include, but are not limited to,ethylene or vinyl, allyl, -1-butenyl, -2-butenyl, -isobutylenyl,-1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, and-2,3-dimethyl-2-butenyl. Examples of alkynyl groups include, but are notlimited to, acetylenic, propargyl, acetylenyl, propynyl, -1-butynyl,-2-butynyl, -1-pentynyl, -2-pentynyl, and -3-methyl-1 butynyl.

Alkenyl and alkynyl groups, whether alone or as part of another group,can be optionally substituted with one or more groups, preferably 1 to 3groups (and any additional substituents selected from halogen),including but not limited to, -halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈alkenyl), —O—(C₂-C₈ alkynyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′,—C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′, —S(O)₂R′,—S(O)R′, —OH, ═O, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN, where each R′ isindependently selected from —H, —C₁-C₈ alkyl, —C₂-C₈ alkyenl, —C₂-C₈alkynyl, or -aryl and wherein said —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl),—O—(C₂-C₈ alkynyl), -aryl, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, and —C₂-C₈alkynyl groups can be optionally further substituted with one or moresubstituents including, but not limited to, —C₁-C₈ alkyl, —C₂-C₈alkenyl, —C₂-C₈ alkynyl, -halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl),—O—(C₂-C₈ alkynyl), -aryl, —C(O)R″, —OC(O)R″, —C(O)OR″, —C(O)NH₂,—C(O)NHR″, —C(O)N(R″)₂, —NHC(O)R″, —SR″, —SO₃R″, —S(O)₂R″, —S(O)R″, —OH,—N₃, —NH₂, —NH(R″), —N(R″)₂ and —CN, where each R″ is independentlyselected from —H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or-aryl.

Unless otherwise noted, the term “alkylene” refers to a saturatedbranched or straight chain hydrocarbon radical having from about 1 toabout 20 carbon atoms (and all combinations and subcombinations ofranges and specific numbers of carbon atoms therein), with from about 1to about 8 carbon atoms being preferred and having two monovalentradical centers derived by the removal of two hydrogen atoms from thesame or two different carbon atoms of a parent alkane. Typical alkylenesinclude, but are not limited to, methylene, ethylene, propylene,butylene, pentylene, hexylene, heptylene, ocytylene, nonylene, decalene,1,4-cyclohexylene, and the like. Alkylene groups, whether alone or aspart of another group, can be optionally substituted with one or moregroups, preferably 1 to 3 groups (and any additional substituentsselected from halogen), including, but not limited to, -halogen,—O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl), -aryl,—C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂,—NHC(O)R′, —SR′, —SO₃R′, —S(O)₂R′, —S(O)R′, —OH, ═O, —N₃, —NH₂, —NH(R′),—N(R′)₂ and —CN, where each R′ is independently selected from —H, —C₁-C₈alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or -aryl and wherein said—O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl), -aryl, —C₁-C₈alkyl, —C₂-C₈ alkenyl, and —C₂-C₈ alkynyl groups can be furtheroptionally substituted with one or more substituents including, but notlimited to, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, -halogen,—O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl), -aryl,—C(O)R″, —OC(O)R″, —C(O)OR″, —C(O)NH₂, —C(O)NHR″, —C(O)N(R″)₂,—NHC(O)R″, —SR″, —SO₃R″, —S(O)₂R″, —S(O)R″, —OH, —N₃, —NH₂, —NH(R″),—N(R″)₂ and —CN, where each R″ is independently selected from —H, —C₁-C₈alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or -aryl.

Unless otherwise noted, the term “alkenylene” refers to an optionallysubstituted alkylene group containing at least one carbon-carbon doublebond. Exemplary alkenylene groups include, for example, ethenylene(—CH═CH—) and propenylene (—CH═CHCH₂—).

Unless otherwise noted, the term “alkynylene” refers to an optionallysubstituted alkylene group containing at least one carbon-carbon triplebond. Exemplary alkynylene groups include, for example, acetylene(—C≡C—), propargyl (—CH₂C≡C—), and 4-pentynyl (—CH₂CH₂CH₂C≡CH—).

Unless otherwise noted, the term “aryl” refers to a monovalent aromatichydrocarbon radical of 6-20 carbon atoms (and all combinations andsubcombinations of ranges and specific numbers of carbon atoms therein)derived by the removal of one hydrogen atom from a single carbon atom ofa parent aromatic ring system. Some aryl groups are represented in theexemplary structures as “Ar”. Typical aryl groups include, but are notlimited to, radicals derived from benzene, substituted benzene, phenyl,naphthalene, anthracene, biphenyl, and the like.

An aryl group, whether alone or as part of another group, can beoptionally substituted with one or more, preferably 1 to 5, or even 1 to2 groups including, but not limited to, -halogen, —C₁-C₈ alkyl, —C₂-C₈alkenyl, —C₂-C₈ alkynyl, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈alkynyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′, —S(O)₂R′, —S(O)R′, —OH, —NO₂, —N₃,—NH₂, —NH(R′), —N(R′)₂ and —CN, where each R′ is independently selectedfrom —H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or -aryl andwherein said —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, O—(C₁-C₈alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl), and -aryl groups can befurther optionally substituted with one or more substituents including,but not limited to, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl,-halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl),-aryl, —C(O)R″, —OC(O)R″, —C(O)OR″, —C(O)NH₂, —C(O)NHR″, —C(O)N(R″)₂,—NHC(O)R″, —SR″, —SO₃R″, —S(O)₂R″, —S(O)R″, —OH, —N₃, —NH₂, —NH(R″),—N(R″)₂ and —CN, where each R″ is independently selected from —H, —C₁-C₈alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or -aryl.

Unless otherwise noted, the term “arylene” refers to an optionallysubstituted aryl group which is divalent (i.e., derived by the removalof two hydrogen atoms from the same or two different carbon atoms of aparent aromatic ring system) and can be in the ortho, meta, or paraconfigurations as shown in the following structures with phenyl as theexemplary aryl group:

typical “—(C₁-C₈ alkylene)aryl,” “—(C₂-C₈ alkenylene)aryl”, “and —(C₂-C₈alkynylene)aryl” groups include, but are not limited to, benzyl,2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl,2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl,2-naphthophenylethan-1-yl and the like.

Unless otherwise noted, the term “heterocycle,” refers to a monocyclic,bicyclic, or polycyclic ring system having from 3 to 14 ring atoms (alsoreferred to as ring members) wherein at least one ring atom in at leastone ring is a heteroatom selected from N, O, P, or S (and allcombinations and subcombinations of ranges and specific numbers ofcarbon atoms and heteroatoms therein). The heterocycle can have from 1to 4 ring heteroatoms independently selected from N, O, P, or S. One ormore N, C, or S atoms in a heterocycle can be oxidized. A monocylicheterocycle preferably has 3 to 7 ring members (e.g., 2 to 6 carbonatoms and 1 to 3 heteroatoms independently selected from N, O, P, or S),and a bicyclic heterocycle preferably has 5 to 10 ring members (e.g., 4to 9 carbon atoms and 1 to 3 heteroatoms independently selected from N,O, P, or S). The ring that includes the heteroatom can be aromatic ornon-aromatic. Unless otherwise noted, the heterocycle is attached to itspendant group at any heteroatom or carbon atom that results in a stablestructure.

Heterocycles are described in Paquette, “Principles of ModernHeterocyclic Chemistry” (W. A. Benjamin, New York, 1968), particularlyChapters 1, 3, 4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds,A series of Monographs” (John Wiley & Sons, New York, 1950 to present),in particular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc.82:5566 (1960).

Examples of “heterocycle” groups include by way of example and notlimitation pyridyl, dihydropyridyl, tetrahydropyridyl (piperidyl),thiazolyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl,imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl, indolyl,indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl, piperidinyl,4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, pyrrolinyl,tetrahydrofuranyl, bis-tetrahydrofuranyl, tetrahydropyranyl,bis-tetrahydropyranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, octahydroisoquinolinyl, azocinyl, triazinyl,6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl, thienyl, thianthrenyl,pyranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxathinyl,2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl,indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl,4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl,quinazolinyl, cinnolinyl, pteridinyl, 4H-carbazolyl, carbazolyl,β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl,chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl,piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl,oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl,and isatinoyl. Preferred “heterocycle” groups include, but are notlimited to, benzofuranyl, benzothiophenyl, indolyl, benzopyrazolyl,coumarinyl, isoquinolinyl, pyrrolyl, thiophenyl, furanyl, thiazolyl,imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl,pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl andtetrazolyl.

A heterocycle group, whether alone or as part of another group, can beoptionally substituted with one or more groups, preferably 1 to 2groups, including but not limited to, —C₁-C₈ alkyl, —C₂-C₈ alkenyl,—C₂-C₈ alkynyl, -halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl),—O—(C₂-C₈ alkynyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′, —S(O)₂R′, —S(O)R′, —OH,—N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN, where each R′ is independentlyselected from —H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or -aryland wherein said —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈alkynyl), —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, and -aryl groupscan be further optionally substituted with one or more substituentsincluding, but not limited to, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈alkynyl, -halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈alkynyl), -aryl, —C(O)R″, —OC(O)R″, —C(O)OR″, —C(O)NH₂, —C(O)NHR″,—C(O)N(R″)₂, —NHC(O)R″, —SR″, —SO₃R″, —S(O)₂R″, —S(O)R″, —OH, —N₃, —NH₂,—NH(R″), —N(R″)₂ and —CN, where each R″ is independently selected from—H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or aryl.

By way of example and not limitation, carbon-bonded heterocycles can bebonded at the following positions: position 2, 3, 4, 5, or 6 of apyridine; position 3, 4, 5, or 6 of a pyridazine; position 2, 4, 5, or 6of a pyrimidine; position 2, 3, 5, or 6 of a pyrazine; position 2, 3, 4,or 5 of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole ortetrahydropyrrole; position 2, 4, or 5 of an oxazole, imidazole orthiazole; position 3, 4, or 5 of an isoxazole, pyrazole, or isothiazole;position 2 or 3 of an aziridine; position 2, 3, or 4 of an azetidine;position 2, 3, 4, 5, 6, 7, or 8 of a quinoline; or position 1, 3, 4, 5,6, 7, or 8 of an isoquinoline. Still more typically, carbon bondedheterocycles include 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl,6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl,2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl,3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or5-thiazolyl.

By way of example and not limitation, nitrogen bonded heterocycles canbe bonded at position 1 of an aziridine, azetidine, pyrrole,pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine,2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline,3-pyrazoline, piperidine, piperazine, indole, indoline, or 1H-indazole;position 2 of a isoindole, or isoindoline; position 4 of a morpholine;and position 9 of a carbazole, or β-carboline. Still more typically,nitrogen bonded heterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl,1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.

Unless otherwise noted, the term “carbocycle,” refers to a saturated orunsaturated non-aromatic monocyclic, bicyclic, or polycyclic ring systemhaving from 3 to 14 ring atoms (and all combinations and subcombinationsof ranges and specific numbers of carbon atoms therein) wherein all ofthe ring atoms are carbon atoms. Monocyclic carbocycles preferably have3 to 6 ring atoms, still more preferably 5 or 6 ring atoms. Bicycliccarbocycles preferably have 7 to 12 ring atoms, e.g., arranged as abicyclo [4,5], [5,5], [5,6] or [6,6] system, or 9 or 10 ring atomsarranged as a bicyclo [5,6] or [6,6] system. The term “carbocycle”includes, for example, a monocyclic carbocycle ring fused to an arylring (e.g., a monocyclic carbocycle ring fused to a benzene ring).Carbocyles preferably have 3 to 8 carbon ring atoms.

Carbocycle groups, whether alone or as part of another group, can beoptionally substituted with, for example, one or more groups, preferably1 or 2 groups (and any additional substituents selected from halogen),including, but not limited to, -halogen, —C₁-C₈ alkyl, —C₂-C₈ alkenyl,—C₂-C₈ alkynyl, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈alkynyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′, —S(O)₂R′, —S(O)R′, —OH, ═O, —N₃,—NH₂, —NH(R′), —N(R′)₂ and —CN, where each R′ is independently selectedfrom —H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or -aryl andwherein said —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, —O—(C₁-C₈alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl), and -aryl groups can befurther optionally substituted with one or more substituents including,but not limited to, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl,-halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl),-aryl, —C(O)R″, —OC(O)R″, —C(O)OR″, —C(O)NH₂, —C(O)NHR″, —C(O)N(R″)₂,—NHC(O)R″, —SR″, —SO₃R″, —S(O)₂R″, —S(O)R″, —OH, —N₃, —NH₂, —NH(R″),—N(R″)₂ and —CN, where each R″ is independently selected from —H, —C₁-C₈alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or -aryl.

Examples of monocyclic carbocylic substituents include -cyclopropyl,-cyclobutyl, -cyclopentyl, -1-cyclopent-1-enyl, -1-cyclopent-2-enyl,-1-cyclopent-3-enyl, cyclohexyl, -1-cyclohex-1-enyl, -1-cyclohex-2-enyl,-1-cyclohex-3-enyl, -cycloheptyl, -cyclooctyl. -1,3-cyclohexadienyl,-1,4-cyclohexadienyl, -1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl,and -cyclooctadienyl.

A “carbocyclo,” whether used alone or as part of another group, refersto an optionally substituted carbocycle group as defined above that isdivalent (i.e., derived by the removal of two hydrogen atoms from thesame or two different carbon atoms of a parent carbocyclic ring system).

Unless otherwise indicated by context, a hyphen (-) designates the pointof attachment to the pendant molecule. Accordingly, the term “—(C₁-C₈alkylene)aryl” or “—C₁-C₈ alkylene(aryl)” refers to a C₁-C₈ alkyleneradical as defined herein wherein the alkylene radical is attached tothe pendant molecule at any of the carbon atoms of the alkylene radicaland one of the hydrogen atoms bonded to a carbon atom of the alkyleneradical is replaced with an aryl radical as defined herein.

When a particular group is “substituted”, that group may have one ormore substituents, preferably from one to five substituents, morepreferably from one to three substituents, most preferably from one totwo substituents, independently selected from the list of substituents.The group can, however, generally have any number of substituentsselected from halogen. Groups that are substituted are so indicated.

It is intended that the definition of any substituent or variable at aparticular location in a molecule be independent of its definitionselsewhere in that molecule. It is understood that substituents andsubstitution patterns on the compounds of this invention can be selectedby one of ordinary skill in the art to provide compounds that arechemically stable and that can be readily synthesized by techniquesknown in the art as well as those methods set forth herein.

Protective groups as used herein refer to groups which selectivelyblock, either temporarily or permanently, one reactive site in amultifunctional compound. Suitable hydroxy-protecting groups for use inthe present invention are pharmaceutically acceptable and may or may notneed to be cleaved from the parent compound after administration to asubject in order for the compound to be active. Cleavage is throughnormal metabolic processes within the body. Hydroxy protecting groupsare well known in the art, see, Protective Groups in Organic Synthesisby T. W. Greene and P. G. M. Wuts (John Wiley & sons, 3^(rd) Edition)incorporated herein by reference in its entirety and for all purposesand include, for example, ether (e.g., alkyl ethers and silyl ethersincluding, for example, dialkylsilylether, trialkylsilylether,dialkylalkoxysilylether), ester, carbonate, carbamates, sulfonate, andphosphate protecting groups. Examples of hydroxy protecting groupsinclude, but are not limited to, methyl ether; methoxymethyl ether,methylthiomethyl ether, (phenyldimethylsilyl)methoxymethyl ether,benzyloxymethyl ether, p-methoxybenzyloxymethyl ether,p-nitrobenzyloxymethyl ether, o-nitrobenzyloxymethyl ether,(4-methoxyphenoxy)methyl ether, guaiacolmethyl ether, t-butoxymethylether, 4-pentenyloxymethyl ether, siloxymethyl ether,2-methoxyethoxymethyl ether, 2,2,2-trichloroethoxymethyl ether,bis(2-chloroethoxy)methyl ether, 2-(trimethylsilyl)ethoxymethyl ether,menthoxymethyl ether, tetrahydropyranyl ether, 1-methoxycylcohexylether, 4-methoxytetrahydrothiopyranyl ether,4-methoxytetrahydrothiopyranyl ether S,S-Dioxide,1-[(2-choro-4-methyl)phenyl]-4-methoxypiperidin-4-yl ether,1-(2-fluorophneyl)-4-methoxypiperidin-4-yl ether, 1,4-dioxan-2-yl ether,tetrahydrofuranyl ether, tetrahydrothiofuranyl ether; substituted ethylethers such as 1-ethoxyethyl ether, 1-(2-chloroethoxy)ethyl ether,1-[2-(trimethylsilyl)ethoxy]ethyl ether, 1-methyl-1-methoxyethyl ether,1-methyl-1-benzyloxyethyl ether, 1-methyl-1-benzyloxy-2-fluoroethylether, 1-methyl-lphenoxyethyl ether, 2-trimethylsilyl ether, t-butylether, allyl ether, propargyl ethers, p-chlorophenyl ether,p-methoxyphenyl ether, benzyl ether, p-methoxybenzyl ether3,4-dimethoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether,tripropylsilylether, dimethylisopropylsilyl ether, diethylisopropylsilylether, dimethylhexylsilyl ether, t-butyldimethylsilyl ether,diphenylmethylsilyl ether, benzoylformate ester, acetate ester,chloroacetate ester, dichloroacetate ester, trichloroacetate ester,trifluoroacetate ester, methoxyacetate ester, triphneylmethoxyacetateester, phenylacetate ester, benzoate ester, alkyl methyl carbonate,alkyl 9-fluorenylmethyl carbonate, alkyl ethyl carbonate, alkyl2,2,2-trichloroethyl carbonate, 1,1-dimethyl-2,2,2-trichloroethylcarbonate, alkylsulfonate, methanesulfonate, benzylsulfonate, tosylate,methylene acetal, ethylidene acetal, and t-butylmethylidene ketal.Preferred protecting groups are represented by the formulas —R^(a),—Si(R^(a))(R^(a))(R^(a)), —C(O)R^(a), —C(O)OR^(a), —C(O)NH(R^(a)),—S(O)₂R^(a), —S(O)₂OH, P(O)(OH)₂, and —P(O)(OH)OR^(a), wherein R^(a) isC₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, —C₁-C₂₀alkylene(carbocycle), —C₂-C₂₀ alkenylene(carbocycle), —C₂-C₂₀alkynylene(carbocycle), —C₆-C₁₀ aryl, —C₁-C₂₀ alkylene(aryl), —C₂-C₂₀alkenylene(aryl), —C₂-C₂₀ alkynylene(aryl), —C₁-C₂₀alkylene(heterocycle), —C₂-C₂₀ alkenylene(heterocycle), or —C₂-C₂₀alkynylene(heterocycle) wherein said alkyl, alkenyl, alkynyl, alkylene,alkenylene, alkynylene, aryl, carbocycle, and heterocycle radicalswhether alone or as part of another group are optionally substituted.

“Altering the native glycosylation pattern” is intended for purposesherein to mean deleting one or more carbohydrate moieties found innative sequence 158P1D7 (either by removing the underlying glycosylationsite or by deleting the glycosylation by chemical and/or enzymaticmeans), and/or adding one or more glycosylation sites that are notpresent in the native sequence 158P1D7. In addition, the phrase includesqualitative changes in the glycosylation of the native proteins,involving a change in the nature and proportions of the variouscarbohydrate moieties present.

The term “analog” refers to a molecule which is structurally similar orshares similar or corresponding attributes with another molecule (e.g. a158P1D7-related protein). For example, an analog of a 158P1D7 proteincan be specifically bound by an antibody or T cell that specificallybinds to 158P1D7.

The term “antibody” is used in the broadest sense unless clearlyindicated otherwise. Therefore, an “antibody” can be naturally occurringor man-made such as monoclonal antibodies produced by conventionalhybridoma technology. 158P1D7 antibodies comprise monoclonal andpolyclonal antibodies as well as fragments containing theantigen-binding domain and/or one or more complementarity determiningregions of these antibodies. As used herein, the term “antibody” refersto any form of antibody or fragment thereof that specifically binds158P1D7 and/or exhibits the desired biological activity and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as theyspecifically bind 158P1D7 and/or exhibit the desired biologicalactivity. Any specific antibody can be used in the methods andcompositions provided herein. Thus, in one embodiment the term“antibody” encompasses a molecule comprising at least one variableregion from a light chain immunoglobulin molecule and at least onevariable region from a heavy chain molecule that in combination form aspecific binding site for the target antigen. In one embodiment, theantibody is an IgG antibody. For example, the antibody is an IgG1, IgG2,IgG3, or IgG4 antibody. The antibodies useful in the present methods andcompositions can be generated in cell culture, in phage, or in variousanimals, including but not limited to cows, rabbits, goats, mice, rats,hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, andapes. Therefore, in one embodiment, an antibody of the present inventionis a mammalian antibody. Phage techniques can be used to isolate aninitial antibody or to generate variants with altered specificity oravidity characteristics. Such techniques are routine and well known inthe art. In one embodiment, the antibody is produced by recombinantmeans known in the art. For example, a recombinant antibody can beproduced by transfecting a host cell with a vector comprising a DNAsequence encoding the antibody. One or more vectors can be used totransfect the DNA sequence expressing at least one VL and one VH regionin the host cell. Exemplary descriptions of recombinant means ofantibody generation and production include Delves, ANTIBODY PRODUCTION:ESSENTIAL TECHNIQUES (Wiley, 1997); Shephard, et al., MONOCLONALANTIBODIES (Oxford University Press, 2000); Goding, MONOCLONALANTIBODIES: PRINCIPLES AND PRACTICE (Academic Press, 1993); and CURRENTPROTOCOLS IN IMMUNOLOGY (John Wiley & Sons, most recent edition). Anantibody of the present invention can be modified by recombinant meansto increase efficacy of the antibody in mediating the desired function.Thus, it is within the scope of the invention that antibodies can bemodified by substitutions using recombinant means. Typically, thesubstitutions will be conservative substitutions. For example, at leastone amino acid in the constant region of the antibody can be replacedwith a different residue. See, e.g., U.S. Pat. Nos. 5,624,821,6,194,551, Application No. WO 9958572; and Angal, et al., Mol. Immunol.30: 105-08 (1993). The modification in amino acids includes deletions,additions, and substitutions of amino acids. In some cases, such changesare made to reduce undesired activities, e.g., complement-dependentcytotoxicity. Frequently, the antibodies are labeled by joining, eithercovalently or non-covalently, a substance which provides for adetectable signal. A wide variety of labels and conjugation techniquesare known and are reported extensively in both the scientific and patentliterature. These antibodies can be screened for binding to normal ordefective 158P1D7. See e.g., ANTIBODY ENGINEERING: A PRACTICAL APPROACH(Oxford University Press, 1996). Suitable antibodies with the desiredbiologic activities can be identified using the following in vitroassays including but not limited to: proliferation, migration, adhesion,soft agar growth, angiogenesis, cell-cell communication, apoptosis,transport, signal transduction, and the following in vivo assays such asthe inhibition of tumor growth. The antibodies provided herein can alsobe useful in diagnostic applications. As capture or non-neutralizingantibodies, they can be screened for the ability to bind to the specificantigen without inhibiting the receptor-binding or biological activityof the antigen. As neutralizing antibodies, the antibodies can be usefulin competitive binding assays. They can also be used to quantify the158P1D7 or its receptor.

The term “antigen-binding portion” or “antibody fragment” of an antibody(or simply “antibody portion”), as used herein, refers to one or morefragments of a 158P1D7 antibody that retain the ability to specificallybind to an antigen (e.g., 158P1D7 and variants; FIG. 1 ). It has beenshown that the antigen-binding function of an antibody can be performedby fragments of a full-length antibody. Examples of binding fragmentsencompassed within the term “antigen-binding portion” of an antibodyinclude (i) a Fab fragment, a monovalent fragment consisting of theV_(L), V_(H), C_(L) and C_(H1) domains; (ii) a F(ab′)₂ fragment, abivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region; (iii) a Fd fragment consisting of the V_(H)and C_(H1) domains; (iv) a Fv fragment consisting of the V_(L) and V_(H)domains of a single arm of an antibody, (v) a dAb fragment (Ward et al.,(1989) Nature 341:544-546), which consists of a V_(H) domain; and (vi)an isolated complementarily determining region (CDR). Furthermore,although the two domains of the Fv fragment, V_(L) and V_(H), are codedfor by separate genes, they can be joined, using recombinant methods, bya synthetic linker that enables them to be made as a single proteinchain in which the V_(L) and V_(H) regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988)Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.These antibody fragments are obtained using conventional techniquesknown to those with skill in the art, and the fragments are screened forutility in the same manner as are intact antibodies.

As used herein, any form of the “antigen” can be used to generate anantibody that is specific for 158P1D7. Thus, the eliciting antigen maybe a single epitope, multiple epitopes, or the entire protein alone orin combination with one or more immunogenicity enhancing agents known inthe art. The eliciting antigen may be an isolated full-length protein, acell surface protein (e.g., immunizing with cells transfected with atleast a portion of the antigen), or a soluble protein (e.g., immunizingwith only the extracellular domain portion of the protein). The antigenmay be produced in a genetically modified cell. The DNA encoding theantigen may be genomic or non-genomic (e.g., cDNA) and encodes at leasta portion of the extracellular domain. As used herein, the term“portion” refers to the minimal number of amino acids or nucleic acids,as appropriate, to constitute an immunogenic epitope of the antigen ofinterest. Any genetic vectors suitable for transformation of the cellsof interest may be employed, including but not limited to adenoviralvectors, plasmids, and non-viral vectors, such as cationic lipids. Inone embodiment, the antibody of the methods and compositions hereinspecifically bind at least a portion of the extracellular domain of the158P1D7 of interest.

The antibodies or antigen binding fragments thereof provided herein maybe conjugated to a “bioactive agent.” As used herein, the term“bioactive agent” refers to any synthetic or naturally occurringcompound that binds the antigen and/or enhances or mediates a desiredbiological effect to enhance cell-killing toxins. In one embodiment, thebinding fragments useful in the present invention are biologicallyactive fragments. As used herein, the term “biologically active” refersto an antibody or antibody fragment that is capable of binding thedesired antigenic epitope and directly or indirectly exerting a biologiceffect. Direct effects include, but are not limited to the modulation,stimulation, and/or inhibition of a growth signal, the modulation,stimulation, and/or inhibition of an anti-apoptotic signal, themodulation, stimulation, and/or inhibition of an apoptotic or necroticsignal, modulation, stimulation, and/or inhibition the ADCC cascade, andmodulation, stimulation, and/or inhibition the CDC cascade.

“Bispecific” antibodies are also useful in the present methods andcompositions. As used herein, the term “bispecific antibody” refers toan antibody, typically a monoclonal antibody, having bindingspecificities for at least two different antigenic epitopes. In oneembodiment, the epitopes are from the same antigen. In anotherembodiment, the epitopes are from two different antigens. Methods formaking bispecific antibodies are known in the art. For example,bispecific antibodies can be produced recombinantly using theco-expression of two immunoglobulin heavy chain/light chain pairs. See,e.g., Milstein et al., Nature 305:537-39 (1983). Alternatively,bispecific antibodies can be prepared using chemical linkage. See, e.g.,Brennan, et al., Science 229:81 (1985). Bispecific antibodies includebispecific antibody fragments. See, e.g., Hollinger, et al., Proc. Natl.Acad. Sci. U.S.A. 90:6444-48 (1993), Gruber, et al., J. Immunol.152:5368 (1994).

The monoclonal antibodies described herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they specifically bindthe target antigen and/or exhibit the desired biological activity (U.S.Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

The term “Chemotherapeutic Agent” refers to all chemical compounds thatare effective in inhibiting tumor growth. Non-limiting examples ofchemotherapeutic agents include alkylating agents; for example, nitrogenmustards, ethyleneimine compounds and alkyl sulphonates;antimetabolites, for example, folic acid, purine or pyrimidineantagonists; mitotic inhibitors, for example, anti-tubulin agents suchas vinca alkaloids, auristatins and derivatives of podophyllotoxin;cytotoxic antibiotics; compounds that damage or interfere with DNAexpression or replication, for example, DNA minor groove binders; andgrowth factor receptor antagonists. In addition, chemotherapeutic agentsinclude cytotoxic agents (as defined herein), antibodies, biologicalmolecules and small molecules.

The term “compound” refers to and encompasses the chemical compounditself as well as, whether explicitly stated or not, and unless thecontext makes clear that the following are to be excluded: amorphous andcrystalline forms of the compound, including polymorphic forms, wherethese forms may be part of a mixture or in isolation; free acid and freebase forms of the compound, which are typically the forms shown in thestructures provided herein; isomers of the compound, which refers tooptical isomers, and tautomeric isomers, where optical isomers includeenantiomers and diastereomers, chiral isomers and non-chiral isomers,and the optical isomers include isolated optical isomers as well asmixtures of optical isomers including racemic and non-racemic mixtures;where an isomer may be in isolated form or in a mixture with one or moreother isomers; isotopes of the compound, including deuterium- andtritium-containing compounds, and including compounds containingradioisotopes, including therapeutically- and diagnostically-effectiveradioisotopes; multimeric forms of the compound, including dimeric,trimeric, etc. forms; salts of the compound, preferably pharmaceuticallyacceptable salts, including acid addition salts and base addition salts,including salts having organic counterions and inorganic counterions,and including zwitterionic forms, where if a compound is associated withtwo or more counterions, the two or more counterions may be the same ordifferent; and solvates of the compound, including hemisolvates,monosolvates, disolvates, etc., including organic solvates and inorganicsolvates, said inorganic solvates including hydrates; where if acompound is associated with two or more solvent molecules, the two ormore solvent molecules may be the same or different. In some instances,reference made herein to a compound of the invention will include anexplicit reference to one or of the above forms, e.g., salts and/orsolvates; however, this reference is for emphasis only, and is not to beconstrued as excluding other of the above forms as identified above.

The terms “complementarity determining region,” and “CDR,” are known inthe art to refer to non-contiguous sequences of amino acids withinantibody variable regions, which confer antigen specificity and bindingaffinity. In general, there are three (3) CDRs in each heavy chainvariable region (CDR-H1, CDR-H2, CDR-H3) and three (3) CDRs in eachlight chain variable region (CDR-L1, CDR-L2, CDR-L3).

The precise amino acid sequence boundaries of a given CDR can be readilydetermined using any of a number of well-known schemes, including thosedescribed by Kabat et al. (1991), “Sequences of Proteins ofImmunological Interest,” 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, MD (“Kabat” numbering scheme),Al-Lazikani et al., (1997) JMB 273,927-948 (“Chothia” numbering scheme),MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigeninteractions: Contact analysis and binding site topography,” J. Mol.Biol. 262, 732-745.” (Contact” numbering scheme), Lefranc M P et al.,“IMGT unique numbering for immunoglobulin and T cell receptor variabledomains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003January; 27(1):55-77 (“IMGT” numbering scheme), and Honegger A andPlückthun A, “Yet another numbering scheme for immunoglobulin variabledomains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun.8; 309(3):657-70, (AHo numbering scheme).

The boundaries of a given CDR may vary depending on the scheme used foridentification. For example, the Kabat scheme is based structuralalignments, while the Chothia scheme is based on structural information.Numbering for both the Kabat and Chothia schemes is based upon the mostcommon antibody region sequence lengths, with insertions accommodated byinsertion letters, for example, “30a,” and deletions appearing in someantibodies. The two schemes place certain insertions and deletions(“indels”) at different positions, resulting in differential numbering.The Contact scheme is based on analysis of complex crystal structuresand is similar in many respects to the Chothia numbering scheme. TableV, infra, lists the positions of CDR-L1, CDR-L2, CDR-L3 and CDR-H1,CDR-H2, CDR-H3 as identified by the Kabat, Chothia, and Contact schemes,respectively. For CDR-H1, residue numbering is given listed using boththe Kabat and Chothia numbering schemes.

Thus, unless otherwise specified, the terms “CDR” and “complementarydetermining region” of a given antibody or region thereof, such as avariable region, as well as individual CDRs (e.g., “CDR-H1, CDR-H2) ofthe antibody or region thereof, should be understood to encompass thecomplementary determining region as defined by any of the known schemesdescribed herein above. In some instances, the scheme for identificationof a particular CDR or CDRs is specified, such as the CDR as defined bythe Kabat, Chothia, or Contact method. In other cases, the particularamino acid sequence of a CDR is given.

As used herein, the term “conservative substitution” refers tosubstitutions of amino acids are known to those of skill in this art andmay be made generally without altering the biological activity of theresulting molecule. Those of skill in this art recognize that, ingeneral, single amino acid substitutions in non-essential regions of apolypeptide do not substantially alter biological activity (see, e.g.,Watson, et al., MOLECULAR BIOLOGY OF THE GENE, The Benjamin/CummingsPub. Co., p. 224 (4th Edition 1987)). Such exemplary substitutions arepreferably made in accordance with those set forth in Table II andTable(s) III(a-b). For example, such changes include substituting any ofisoleucine (I), valine (V), and leucine (L) for any other of thesehydrophobic amino acids; aspartic acid (D) for glutamic acid (E) andvice versa; glutamine (Q) for asparagine (N) and vice versa; and serine(S) for threonine (T) and vice versa. Other substitutions can also beconsidered conservative, depending on the environment of the particularamino acid and its role in the three-dimensional structure of theprotein. For example, glycine (G) and alanine (A) can frequently beinterchangeable, as can alanine (A) and valine (V). Methionine (M),which is relatively hydrophobic, can frequently be interchanged withleucine and isoleucine, and sometimes with valine. Lysine (K) andarginine (R) are frequently interchangeable in locations in which thesignificant feature of the amino acid residue is its charge and thediffering pK's of these two amino acid residues are not significant.Still other changes can be considered “conservative” in particularenvironments (see, e.g. Table III(a) herein; pages 13-15 “Biochemistry”2nd ED. Lubert Stryer ed (Stanford University); Henikoff et al, PNAS1992 Vol 89 10915-10919; Lei et al., J Biol Chem 1995 May 19;270(20):11882-6). Other substitutions are also permissible and may bedetermined empirically or in accord with known conservativesubstitutions.

The term “cytotoxic agent” refers to a substance that inhibits orprevents the expression activity of cells, function of cells and/orcauses destruction of cells. The term is intended to include radioactiveisotopes, chemotherapeutic agents, and toxins such as small moleculetoxins or enzymatically active toxins of bacterial, fungal, plant oranimal origin, including fragments and/or variants thereof. Examples ofcytotoxic agents include, but are not limited to auristatins (e.g.,auristatin E, auristatin F, MMAE and MMAF), auromycins, maytansinoids,ricin, ricin A-chain, combrestatin, duocarmycins, dolastatins,doxorubicin, daunorubicin, taxols, cisplatin, cc1065, ethidium bromide,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine,dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonasexotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain,alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin,curicin, crotin, calicheamicin, Sapaonaria officinalis inhibitor, andglucocorticoid and other chemotherapeutic agents, as well asradioisotopes such as At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹²or ²¹³, P³² and radioactive isotopes of Lu including Lu¹⁷⁷. Antibodiesmay also be conjugated to an anti-cancer pro-drug activating enzymecapable of converting the pro-drug to its active form.

As used herein, the term “diabodies” refers to small antibody fragmentswith two antigen-binding sites, which fragments comprise a heavy chainvariable domain (V_(H)) connected to a light chain variable domain(V_(L)) in the same polypeptide chain (V_(H)-V_(L)). By using a linkerthat is too short to allow pairing between the two domains on the samechain, the domains are forced to pair with the complementary domains ofanother chain and create two antigen-binding sites. Diabodies aredescribed more fully in, e.g., EP 404,097; WO 93/11161; and Hollinger etal., Proc. Natl. Acad. Sci. USA 90:6444-48 (1993).

The term “deplete,” in the context of the effect of a 158P1D7 bindingagent on 158P1D7-expressing cells, refers to a reduction in the numberof or elimination of the 158P1D7-expressing cells.

The term “gene product” is used herein to indicate a peptide/protein ormRNA. For example, a “gene product of the invention” is sometimesreferred to herein as a “cancer amino acid sequence”, “cancer protein”,“protein of a cancer listed in Table I”, a “cancer mRNA”, “mRNA of acancer listed in Table I”, etc. In one embodiment, the cancer protein isencoded by a nucleic acid of FIG. 1 . The cancer protein can be afragment, or alternatively, be the full-length protein encoded bynucleic acids of FIG. 1 . In one embodiment, a cancer amino acidsequence is used to determine sequence identity or similarity. Inanother embodiment, the sequences are naturally occurring allelicvariants of a protein encoded by a nucleic acid of FIG. 1 . In anotherembodiment, the sequences are sequence variants as further describedherein.

“Heteroconjugate” antibodies are useful in the present methods andcompositions. As used herein, the term “heteroconjugate antibody” refersto two covalently joined antibodies. Such antibodies can be preparedusing known methods in synthetic protein chemistry, including usingcrosslinking agents. See, e.g., U.S. Pat. No. 4,676,980.

The term “homolog” refers to a molecule which exhibits homology toanother molecule, by for example, having sequences of chemical residuesthat are the same or similar at corresponding positions.

In one embodiment, the antibody provided herein is a “human antibody.”As used herein, the term “human antibody” refers to an antibody in whichessentially the entire sequences of the light chain and heavy chainsequences, including the complementary determining regions (CDRs), arefrom human genes. In one embodiment, human monoclonal antibodies areprepared by the trioma technique, the human B-cell technique (see, e.g.,Kozbor, et al., Immunol. Today 4: 72 (1983), EBV transformationtechnique (see, e.g., Cole et al. MONOCLONAL ANTIBODIES AND CANCERTHERAPY 77-96 (1985)), or using phage display (see, e.g., Marks et al.,J. Mol. Biol. 222:581 (1991)). In a specific embodiment, the humanantibody is generated in a transgenic mouse. Techniques for making suchpartially to fully human antibodies are known in the art and any suchtechniques can be used. According to one particularly preferredembodiment, fully human antibody sequences are made in a transgenicmouse engineered to express human heavy and light chain antibody genes.An exemplary description of preparing transgenic mice that produce humanantibodies found in Application No. WO 02/43478 and U.S. Pat. No.6,657,103 (Abgenix) and its progeny. B cells from transgenic mice thatproduce the desired antibody can then be fused to make hybridoma celllines for continuous production of the antibody. See, e.g., U.S. Pat.Nos. 5,569,825; 5,625,126; 5,633,425; 5,661,016; and 5,545,806; andJakobovits, Adv. Drug Del. Rev. 31:33-42 (1998); Green, et al., J. Exp.Med. 188:483-95 (1998).

As used herein, the term “humanized antibody” refers to forms ofantibodies that contain sequences from non-human (e.g., murine)antibodies as well as human antibodies. Such antibodies are chimericantibodies which contain minimal sequence derived from non-humanimmunoglobulin. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin sequence. Thehumanized antibody optionally also will comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. See e.g., Cabilly U.S. Pat. No. 4,816,567; Queen et al.(1989) Proc. Nat'l Acad. Sci. USA 86:10029-10033; and ANTIBODYENGINEERING: A PRACTICAL APPROACH (Oxford University Press 1996).

The terms “inhibit” or “inhibition of” as used herein means to reduce bya measurable amount, or to prevent entirely.

The phrases “isolated” or “biologically pure” refer to material which issubstantially or essentially free from components which normallyaccompany the material as it is found in its native state. Thus,isolated peptides in accordance with the invention preferably do notcontain materials normally associated with the peptides in their in situenvironment. For example, a polynucleotide is said to be “isolated” whenit is substantially separated from contaminant polynucleotides thatcorrespond or are complementary to genes other than the 158P1D7 genes orthat encode polypeptides other than 158P1D7 gene product or fragmentsthereof. A skilled artisan can readily employ nucleic acid isolationprocedures to obtain an isolated 158P1D7 polynucleotide. A protein issaid to be “isolated,” for example, when physical, mechanical orchemical methods are employed to remove the 158P1D7 proteins fromcellular constituents that are normally associated with the protein. Askilled artisan can readily employ standard purification methods toobtain an isolated 158P1D7 protein. Alternatively, an isolated proteincan be prepared by chemical means.

Suitable “labels” include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent moieties, chemiluminescent moieties, magneticparticles, and the like. Patents teaching the use of such labels includeU.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;4,275,149; and 4,366,241. In addition, the antibodies provided hereincan be useful as the antigen-binding component of fluorobodies. Seee.g., Zeytun et al., Nat. Biotechnol. 21:1473-79 (2003).

The term “mammal” refers to any organism classified as a mammal,including mice, rats, rabbits, dogs, cats, cows, horses and humans. Inone embodiment of the invention, the mammal is a mouse. In anotherembodiment of the invention, the mammal is a human.

The terms “metastatic cancer” and “metastatic disease” mean cancers thathave spread to regional lymph nodes or to distant sites, and are meantto include stage D disease under the AUA system and stage TxNxM+ underthe TNM system.

The term “modulator” or “test compound” or “drug candidate” orgrammatical equivalents as used herein describe any molecule, e.g.,protein, oligopeptide, small organic molecule, polysaccharide,polynucleotide, etc., to be tested for the capacity to directly orindirectly alter the cancer phenotype or the expression of a cancersequence, e.g., a nucleic acid or protein sequences, or effects ofcancer sequences (e.g., signaling, gene expression, protein interaction,etc.) In one aspect, a modulator will neutralize the effect of a cancerprotein of the invention. By “neutralize” is meant that an activity of aprotein is inhibited or blocked, along with the consequent effect on thecell. In another aspect, a modulator will neutralize the effect of agene, and its corresponding protein, of the invention by normalizinglevels of said protein. In preferred embodiments, modulators alterexpression profiles, or expression profile nucleic acids or proteinsprovided herein, or downstream effector pathways. In one embodiment, themodulator suppresses a cancer phenotype, e.g. to a normal tissuefingerprint. In another embodiment, a modulator induced a cancerphenotype. Generally, a plurality of assay mixtures is run in parallelwith different agent concentrations to obtain a differential response tothe various concentrations. Typically, one of these concentrationsserves as a negative control, i.e., at zero concentration or below thelevel of detection.

Modulators, drug candidates, or test compounds encompass numerouschemical classes, though typically they are organic molecules,preferably small organic compounds having a molecular weight of morethan 100 and less than about 2,500 Daltons. Preferred small moleculesare less than 2000, or less than 1500 or less than 1000 or less than 500D. Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Modulators also comprise biomolecules such aspeptides, saccharides, fatty acids, steroids, purines, pyrimidines,derivatives, structural analogs or combinations thereof. Particularlypreferred are peptides. One class of modulators are peptides, forexample of from about five to about 35 amino acids, with from about fiveto about 20 amino acids being preferred, and from about 7 to about 15being particularly preferred. Preferably, the cancer modulatory proteinis soluble, includes a non-transmembrane region, and/or, has anN-terminal Cys to aid in solubility. In one embodiment, the C-terminusof the fragment is kept as a free acid and the N-terminus is a freeamine to aid in coupling, i.e., to cysteine. In one embodiment, a cancerprotein of the invention is conjugated to an immunogenic agent asdiscussed herein. In one embodiment, the cancer protein is conjugated toBSA. The peptides of the invention, e.g., of preferred lengths, can belinked to each other or to other amino acids to create a longerpeptide/protein. The modulatory peptides can be digests of naturallyoccurring proteins as is outlined above, random peptides, or “biased”random peptides. In a preferred embodiment, peptide/protein-basedmodulators are antibodies, and fragments thereof, as defined herein.

The term “monoclonal antibody”, as used herein, refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic epitope. In contrast, conventional(polyclonal) antibody preparations typically include a multitude ofantibodies directed against (or specific for) different epitopes. In oneembodiment, the polyclonal antibody contains a plurality of monoclonalantibodies with different epitope specificities, affinities, oravidities within a single antigen that contains multiple antigenicepitopes. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al., Nature256: 495 (1975), or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature 352: 624-628 (1991) and Marks et al., J. Mol.Biol. 222: 581-597 (1991), for example. These monoclonal antibodies willusually bind with at least a Kd of about 1 μM, more usually at leastabout 300 nM, typically at least about 30 nM, preferably at least about10 nM, more preferably at least about 3 nM or better, usually determinedby ELISA.

A “pharmaceutical excipient” comprises a material such as an adjuvant, acarrier, pH-adjusting and buffering agents, tonicity adjusting agents,wetting agents, preservative, and the like.

“Pharmaceutically acceptable” refers to a non-toxic, inert, and/orcomposition that is physiologically compatible with humans or othermammals.

The term “polynucleotide” means a polymeric form of nucleotides of atleast 10 bases or base pairs in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide, and ismeant to include single and double stranded forms of DNA and/or RNA. Inthe art, this term if often used interchangeably with “oligonucleotide”.A polynucleotide can comprise a nucleotide sequence disclosed hereinwherein thymidine (T), as shown for example in FIG. 1 , can also beuracil (U); this definition pertains to the differences between thechemical structures of DNA and RNA, in particular the observation thatone of the four major bases in RNA is uracil (U) instead of thymidine(T).

The term “polypeptide” means a polymer of at least about 4, 5, 6, 7, or8 amino acids. Throughout the specification, standard three letter (See,Table III) or single letter designations for amino acids are used. Inthe art, this term is often used interchangeably with “peptide” or“protein”.

A “recombinant” DNA or RNA molecule is a DNA or RNA molecule that hasbeen subjected to molecular manipulation in vitro.

As used herein, the term “single-chain Fv” or “scFv” or “single chain”antibody refers to antibody fragments comprising the V_(H) and V_(L)domains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thesFv to form the desired structure for antigen binding. For a review ofsFv, see Pluckthun, THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113,Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

As used herein, the terms “specific”, “specifically binds” and “bindsspecifically” refer to the selective binding of the antibody to thetarget antigen epitope. Antibodies can be tested for specificity ofbinding by comparing binding to appropriate antigen to binding toirrelevant antigen or antigen mixture under a given set of conditions.If the antibody binds to the appropriate antigen at least 2, 5, 7, andpreferably 10 times more than to irrelevant antigen or antigen mixturethen it is considered to be specific. In one embodiment, a specificantibody is one that only binds the 158P1D7 antigen, but does not bindto the irrelevant antigen. In another embodiment, a specific antibody isone that binds human 158P1D7 antigen but does not bind a non-human158P1D7 antigen with 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or greater amino acid homology with the 158P1D7antigen. In another embodiment, a specific antibody is one that bindshuman 158P1D7 antigen and binds murine 158P1D7 antigen, but with ahigher degree of binding the human antigen. In another embodiment, aspecific antibody is one that binds human 158P1D7 antigen and bindsprimate 158P1D7 antigen, but with a higher degree of binding the humanantigen. In another embodiment, the specific antibody binds to human158P1D7 antigen and any non-human 158P1D7 antigen, but with a higherdegree of binding the human antigen or any combination thereof.

As used herein “to treat” or “therapeutic” and grammatically relatedterms, refer to any improvement of any consequence of disease, such asprolonged survival, less morbidity, and/or a lessening of side effectswhich are the byproducts of an alternative therapeutic modality; as isreadily appreciated in the art, full eradication of disease is apreferred but albeit not a requirement for a treatment act.

The term “variant” refers to a molecule that exhibits a variation from adescribed type or norm, such as a protein that has one or more differentamino acid residues in the corresponding position(s) of a specificallydescribed protein (e.g. the 158P1D7 protein shown in FIG. 1 .) An analogis an example of a variant protein. Splice isoforms and singlenucleotides polymorphisms (SNPs) are further examples of variants.

The “158P1D7 proteins” and/or “158P1D7 related proteins” of theinvention include those specifically identified herein (see, FIG. 1 ),as well as allelic variants, conservative substitution variants, analogsand homologs that can be isolated/generated and characterized withoutundue experimentation following the methods outlined herein or readilyavailable in the art. Fusion proteins that combine parts of different158P1D7 proteins or fragments thereof, as well as fusion proteins of a158P1D7 protein and a heterologous polypeptide are also included. Such158P1D7 proteins are collectively referred to as the 158P1D7-relatedproteins, the proteins of the invention, or 158P1D7. The term“158P1D7-related protein” refers to a polypeptide fragment or a 158P1D7protein sequence of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, or more than 25 amino acids; or, atleast 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 105,110,115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180,185, 190, 195, 200, 225, 250, 275, 300, 325, 330, 335, 345, 355, 365,375, 385, 395, 405, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650,675, 700, 725, 750, 775, 800, 825, 830, 835, 840, 841 or more aminoacids.

II.) 158P1D7 Antibodies

Another aspect of the invention provides antibodies that bind to158P1D7-related proteins (See FIG. 1 ). In one embodiment, the antibodythat binds to 158P1D7-related proteins is an antibody that specificallybinds to 158P1D7 protein comprising amino acid sequence of SEQ ID NO.:2. The antibody that specifically binds to 158P1D7 protein comprisingamino acid sequence of SEQ ID NO.: 2 includes antibodies that can bindto other 158P1 D7-related proteins. For example, antibodies that bind158P1D7 protein comprising amino acid sequence of SEQ ID NO.: 2 can bind158P1D7-related proteins such as 158P1D7 variants and the homologs oranalogs thereof.

158P1D7 antibodies of the invention are particularly useful in cancer(see, e.g., Table I) prognostic assays, imaging, diagnostic, andtherapeutic methodologies. Similarly, such antibodies are useful in thetreatment, and/or prognosis of bladder and other cancers, to the extent158P1D7 is also expressed or overexpressed in these other cancers.Moreover, 158P1D7 antibodies of the invention are therapeutically usefulin treating cancers in which the expression of 158P1D7 is involvedespecially bladder cancer, such as advanced or metastatic bladdercancers when the antibodies are conjugated to monomethyl auristatin E(MMAE) described herein.

Various methods for the preparation of antibodies, specificallymonoclonal antibodies, are well known in the art. For example,antibodies can be prepared by immunizing a suitable mammalian host usinga 158P1D7-related protein, peptide, or fragment, in isolated orimmunoconjugated form (Antibodies: A Laboratory Manual, CSH Press, Eds.,Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press,NY (1989)). In addition, fusion proteins of 158P1D7 can also be used,such as a 158P1D7 GST-fusion protein. In a particular embodiment, a GSTfusion protein comprising all or most of the amino acid sequence of FIG.1 is produced, and then used as an immunogen to generate appropriateantibodies. In another embodiment, a 158P1D7-related protein issynthesized and used as an immunogen.

In addition, naked DNA immunization techniques known in the art are used(with or without purified 158P1D7-related protein or 158P1D7 expressingcells) to generate an immune response to the encoded immunogen (forreview, see Donnelly et al., 1997, Ann. Rev. Immunol. 15: 617-648).

The amino acid sequence of a 158P1D7 protein as shown in FIG. 1 can beanalyzed to select specific regions of the 158P1D7 protein forgenerating antibodies. For example, hydrophobicity and hydrophilicityanalyses of a 158P1D7 amino acid sequence are used to identifyhydrophilic regions in the 158P1D7 structure. Regions of a 158P1D7protein that show immunogenic structure, as well as other regions anddomains, can readily be identified using various other methods known inthe art, such as Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg,Karplus-Schultz or Jameson-Wolf analysis. Hydrophilicity profiles can begenerated using the method of Hopp, T. P. and Woods, K. R., 1981, Proc.Natl. Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can begenerated using the method of Kyte, J. and Doolittle, R. F., 1982, J.Mol. Biol. 157:105-132. Percent (%) Accessible Residues profiles can begenerated using the method of Janin J., 1979, Nature 277:491-492.Average Flexibility profiles can be generated using the method ofBhaskaran R., Ponnuswamy P. K., 1988, Int. J. Pept. Protein Res.32:242-255. Beta-turn profiles can be generated using the method ofDeleage, G., Roux B., 1987, Protein Engineering 1:289-294. Thus, eachregion identified by any of these programs or methods is within thescope of the present invention. Preferred methods for the generation of158P1D7 antibodies are further illustrated by way of the examplesprovided herein. Methods for preparing a protein or polypeptide for useas an immunogen are well known in the art. Also well known in the artare methods for preparing immunogenic conjugates of a protein with acarrier, such as BSA, KLH or other carrier protein. In somecircumstances, direct conjugation using, for example, carbodiimidereagents are used; in other instances linking reagents such as thosesupplied by Pierce Chemical Co., Rockford, IL, are effective.Administration of a 158P1D7 immunogen is often conducted by injectionover a suitable time period and with use of a suitable adjuvant, as isunderstood in the art. During the immunization schedule, titers ofantibodies can be taken to determine adequacy of antibody formation.

158P1D7 monoclonal antibodies can be produced by various means wellknown in the art. For example, immortalized cell lines that secrete adesired monoclonal antibody are prepared using the standard hybridomatechnology of Kohler and Milstein or modifications that immortalizeantibody-producing B cells, as is generally known. Immortalized celllines that secrete the desired antibodies are screened by immunoassay inwhich the antigen is a 158P1D7-related protein. When the appropriateimmortalized cell culture is identified, the cells can be expanded andantibodies produced either from in vitro cultures or from ascites fluid.

The antibodies or fragments of the invention can also be produced byrecombinant means. Regions that bind specifically to the desired regionsof a 158P1D7 protein can also be produced in the context of chimeric orcomplementarity-determining region (CDR) grafted antibodies of multiplespecies origin. Humanized or human 158P1D7 antibodies can also beproduced, and are preferred for use in therapeutic contexts. Methods forhumanizing murine and other non-human antibodies, by substituting one ormore of the non-human antibody CDRs for corresponding human antibodysequences, are well known (see for example, Jones et al., 1986, Nature321: 522-525; Riechmann et al., 1988, Nature 332: 323-327; Verhoeyen etal., 1988, Science 239: 1534-1536). See also, Carter et al., 1993, Proc.Natl. Acad. Sci. USA 89: 4285 and Sims et al., 1993, J. Immunol. 151:2296.

In a preferred embodiment, the antibodies of the present inventioncomprise fully human 158P1D7 antibodies (158P1D7 MAbs). Various methodsin the art provide means for producing fully human 158P1D7 MAbs. Forexample, a preferred embodiment provides for techniques using transgenicmice, inactivated for antibody production, engineered with human heavyand light chains loci referred to as Xenomouse (Amgen Fremont, Inc.). Anexemplary description of preparing transgenic mice that produce humanantibodies can be found in U.S. Pat. No. 6,657,103. See, also, U.S. Pat.Nos. 5,569,825; 5,625,126; 5,633,425; 5,661,016; and 5,545,806; andMendez, et. al. Nature Genetics, 15: 146-156 (1998); Kellerman, S. A. &Green, L. L., Curr. Opin. Biotechnol 13, 593-597 (2002).

In addition, human antibodies of the invention can be generated usingthe HuMAb mouse (Medarex, Inc.) which contains human immunoglobulin geneminiloci that encode unrearranged human heavy (mu and gamma) and kappalight chain immunoglobulin sequences, together with targeted mutationsthat inactivate the endogenous mu and kappa chain loci (see e.g.,Lonberg, et al. (1994) Nature 368(6474): 856-859).

In another embodiment, fully human antibodies of the invention can beraised using a mouse that carries human immunoglobulin sequences ontransgenes and transchromosomes, such as a mouse that carries a humanheavy chain transgene and a human light chain transchromosome. Suchmice, referred to herein as “KM mice”, such mice are described inTomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727 and PCTPublication WO 02/43478 to Tomizuka, et al.

Human monoclonal antibodies of the invention can also be prepared usingphage display methods for screening libraries of human immunoglobulingenes. Such phage display methods for isolating human antibodies areestablished in the art. See for example: U.S. Pat. Nos. 5,223,409;5,403,484; and U.S. Pat. No. 5,571,698 to Ladner et al.; U.S. Pat. Nos.5,427,908 and 5,580,717 to Dower et al.; U.S. Pat. Nos. 5,969,108 and6,172,197 to McCafferty et al.; and U.S. Pat. Nos. 5,885,793; 6,521,404;6,544,731; 6,555,313; 6,582,915 and 6,593,081 to Griffiths et al.

Human monoclonal antibodies of the invention can also be prepared usingSCID mice into which human immune cells have been reconstituted suchthat a human antibody response can be generated upon immunization. Suchmice are described in, for example, U.S. Pat. Nos. 5,476,996 and5,698,767 to Wilson et al.

Human monoclonal antibodies of the invention can also be prepared usingmice into which genomic sequences bearing endogenous mouse variablesegments at the immunoglobulin heavy chain (VH, DH, and JH segments)and/or kappa light chain (VK and JK) loci have been replaced, in wholeor in part, with human genomic sequences bearing unrearranged germlinevariable segments of the human immunoglobulin heavy chain (VH, DH, andJH) and/or kappa light chain (VK and JK) loci (Regeneron, Tarrytown,NY). See, for example, U.S. Pat. Nos. 6,586,251, 6,596,541, 7,105,348,6,528,313, 6,638,768, and 6,528,314.

In one embodiment, an 158P1D7 MAbs of the invention comprises heavy andlight chain variable regions of an antibody designated Ha15-10ac12produced by a Chinese Hamster Ovary (CHO) cell deposited under theAmerican Type Culture Collection (ATCC) Accession No.: PTA-13102 (See,FIG. 3 ), or heavy and light variable regions comprising amino acidsequences that are homologous to the amino acid sequences of the heavyand light chain variable regions of Ha15-10ac12, such as functionalfragments thereof, and wherein the antibodies retain the desiredfunctional properties of the 158P1D7 MAbs of the invention. The heavychain variable region of Ha15-10ac12 has the amino acid sequence rangingfrom the 1^(st) Q residue to the 120^(th) residue of SEQ ID NO: 7, andthe light chain variable region of Ha15-10ac12 has the amino acidsequence ranging from the 1^(st) D residue to the 113^(th) R residue ofSEQ ID NO: 8.

In one embodiment, the 158P1D7 antibody contains a heavy chain CDR ofthe heavy chain variable region of HA15-10ac12, such as heavy chain CDR1, 2, and/or 3 of the heavy chain variable region of HA15-10ac12, e.g.,CDR1, CDR2, and/or CDR3 of the amino acid sequence set forth as SEQ IDNO: 7, determined by any known numbering scheme for identifying CDRs,such as any described herein. In one embodiment, the 158P1D7 antibodycontains a light chain CDR of the light chain variable region ofHA15-10ac12, such as light chain CDR 1, 2, and/or 3 of the light chainvariable region of HA15-10ac12, e.g., CDR1, CDR2, and/or CDR3 of theamino acid sequence set forth as SEQ ID NO: 8, as determined by anyknown numbering scheme for identifying CDRs, such as any describedherein. In one aspect, CDRs 1-3 of the heavy chain variable region ofHa15-10ac12 contain the amino acid sequences ranging from residues31-35, from residues 50-66, and from residues 99-108, respectively, ofSEQ ID NO: 7. In one aspect, CDRs 1-3 of the light chain variable regionof Ha15-10ac12 contain the amino acid sequences ranging from residues24-39, from residues 55-61, and from residues 94-102, respectively, ofSEQ ID NO: 8. Thus, in some aspects, the 158P1D7 antibody contains aheavy chain CDR1 having residues 31-35 of SEQ ID NO: 7, a heavy chainCDR2 having residues 50-66 of SEQ ID NO: 7, and/or a heavy chain CDR3having residues 99-108 of SEQ ID NO: 7 and/or a light chain CDR1 havingresidues 24-39 of SEQ ID NO: 8, a CDR2 having residues 55-61 of SEQ IDNO: 8, and/or a CDR3 having residues 94-102 of SEQ ID NO: 8. Also amongthe provided embodiments are antibodies that compete for binding toantigen with antibodies having such variable region and/or CDRsequences. In some embodiments, the provided antibody includes aconstant region. The constant region can be any subclass of constantregion. In one embodiment, human IgG2 constant region as the heavy chainconstant region and human Ig kappa constant region as the light chainconstant region can be used.

For example, the invention provides an isolated monoclonal antibody, orantigen binding portion thereof, comprising a heavy chain variableregion and a light chain variable region, wherein:

-   -   (a) the heavy chain variable region comprises an amino acid        sequence that is at least 80% homologous to heavy chain variable        region amino acid sequence set forth in FIG. 3 ; and    -   (b) the light chain variable region comprises an amino acid        sequence that is at least 80% homologous to the light chain        variable region amino acid sequence set forth in FIG. 3 .

In other embodiments, the V_(H) and/or V_(L) amino acid sequences may be85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% homologous to the V_(H) and V_(L) sequences set forth in FIG. 3 .

In another embodiment, the invention provides an isolated monoclonalantibody, or antigen binding portion thereof, comprising a humanizedheavy chain variable region and a humanized light chain variable region,wherein:

-   -   (a) the heavy chain variable region comprises one, two, or three        complementarity determining regions (CDRs) having the amino acid        sequence(s) of the heavy chain variable region CDRs 1, 2, and/or        3, of the heavy chain variable region set forth in FIG. 3 , as        determined using any known numbering scheme, such as any        described herein;    -   (b) the light chain variable region comprises one, two, or three        CDRs having the amino acid sequence(s) of the light chain        variable region CDRs 1, 2, and/or 3, of the light chain variable        region set forth in FIG. 3 , as determined using any known        numbering scheme, such as any described herein.

In another embodiment, the antibody or antigen binding portion thereofcompetes for binding with an antibody having such heavy and/or lightchain CDR(s).

Engineered antibodies of the invention include those in whichmodifications have been made to framework residues within V_(H) and/orV_(L) (e.g. to improve the properties of the antibody). Typically suchframework modifications are made to decrease the immunogenicity of theantibody. For example, one approach is to “backmutate” one or moreframework residues to the corresponding germline sequence. Morespecifically, an antibody that has undergone somatic mutation maycontain framework residues that differ from the germline sequence fromwhich the antibody is derived. Such residues can be identified bycomparing the antibody framework sequences to the germline sequencesfrom which the antibody is derived. To return the framework regionsequences to their germline configuration, the somatic mutations can be“backmutated” to the germline sequence by, for example, site-directedmutagenesis or PCR-mediated mutagenesis (e.g., “backmutated” fromleucine to methionine). Such “backmutated” antibodies are also intendedto be encompassed by the invention.

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T-cell epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. PatentPublication No. 2003/0153043 by Carr et al.

In addition or alternative to modifications made within the framework orCDR regions, antibodies of the invention may be engineered to includemodifications within the Fc region, typically to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. Furthermore, a 158P1D7 MAb of the invention maybe chemically modified (e.g., one or more chemical moieties can beattached to the antibody) or be modified to alter its glycosylation,again to alter one or more functional properties of the MAb. Each ofthese embodiments is described in further detail below.

In one embodiment, the hinge region of CH1 is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425 by Bodmer et al. The number of cysteine residues in thehinge region of CH1 is altered to, for example, facilitate assembly ofthe light and heavy chains or to increase or decrease the stability ofthe 158P1D7 MAb.

In another embodiment, the Fc hinge region of an antibody is mutated todecrease the biological half life of the 158P1D7 MAb. More specifically,one or more amino acid mutations are introduced into the CH2-CH3 domaininterface region of the Fc-hinge fragment such that the antibody hasimpaired Staphylococcyl protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745 by Ward et al.

In another embodiment, the 158P1D7 MAb is modified to increase itsbiological half life. Various approaches are possible. For example,mutations can be introduced as described in U.S. Pat. No. 6,277,375 toWard. Alternatively, to increase the biological half life, the antibodycan be altered within the CH1 or CL region to contain a salvage receptorbinding epitope taken from two loops of a CH2 domain of an Fc region ofan IgG, as described in U.S. Pat. Nos. 5,869,046 and 6,121,022 by Prestaet al.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector function(s) of the 158P1D7 MAb. For example, one or more aminoacids selected from amino acid specific residues can be replaced with adifferent amino acid residue such that the antibody has an alteredaffinity for an effector ligand but retains the antigen-binding abilityof the parent antibody. The effector ligand to which affinity is alteredcan be, for example, an Fc receptor or the C1 component of complement.This approach is described in further detail in U.S. Pat. Nos. 5,624,821and 5,648,260, both by Winter et al.

Reactivity of 158P1D7 antibodies with a 158P1D7-related protein can beestablished by a number of well known means, including Western blot,immunoprecipitation, ELISA, and FACS analyses using, as appropriate,158P1D7-related proteins, 158P1D7-expressing cells or extracts thereof.A 158P1D7 antibody or fragment thereof can be labeled with a detectablemarker or conjugated to a second molecule. Suitable detectable markersinclude, but are not limited to, a radioisotope, a fluorescent compound,a bioluminescent compound, chemiluminescent compound, a metal chelatoror an enzyme. Further, bi-specific antibodies specific for two or more158P1D7 epitopes are generated using methods generally known in the art.Homodimeric antibodies can also be generated by cross-linking techniquesknown in the art (e.g., Wolff et al., Cancer Res. 53: 2560-2565).

In yet another preferred embodiment, the 158P1D7 MAb of the invention isan antibody comprising heavy and light chain of an antibody designatedHa15-10ac12. The heavy chain of Ha15-10ac12 consists of the amino acidsequence ranging from 1^(st) Q residue to the 446^(th) K residue of SEQID NO: 7 and the light chain of Ha15-10ac12 consists of amino acidsequence ranging from 1^(st) D residue to the 219^(th) C residue of SEQID NO: 8 sequence. The sequence of which is set forth in FIG. 2 and FIG.3 . In a preferred embodiment, Ha15-10ac12 is conjugated to a cytotoxicagent.

The Chinese Hamster Ovary (CHO) cell producing the antibody designatedHa15-10ac12 was sent (via Federal Express) to the American Type CultureCollection (ATCC), P.O. Box 1549, Manassas, VA 20108 on 25 Jul. 2012 andassigned Accession number PTA-13102.

III.) Antibody-Drug Conjugates Generally

In another aspect, the invention provides antibody-drug conjugates(ADCs), comprising an antibody conjugated to a cytotoxic agent such as achemotherapeutic agent, a drug, a growth inhibitory agent, a toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate). In another aspect, the invention further providesmethods of using the ADCs. In one aspect, an ADC comprises any of theabove 158P1D7 MAbs covalently attached to a cytotoxic agent or adetectable agent.

The use of antibody-drug conjugates for the local delivery of cytotoxicor cytostatic agents, i.e. drugs to kill or inhibit tumor cells in thetreatment of cancer (Syrigos and Epenetos (1999) Anticancer Research19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg Del. Rev.26:151-172; U.S. Pat. No. 4,975,278) allows targeted delivery of thedrug moiety to tumors, and intracellular accumulation therein, wheresystemic administration of these unconjugated drug agents may result inunacceptable levels of toxicity to normal cells as well as the tumorcells sought to be eliminated (Baldwin et al., (1986) Lancet pp. (Mar.15, 1986):603-05; Thorpe, (1985) “Antibody Carriers Of Cytotoxic AgentsIn Cancer Therapy: A Review,” in Monoclonal Antibodies '84: BiologicalAnd Clinical Applications, A. Pinchera et al. (ed.s), pp. 475-506).Maximal efficacy with minimal toxicity is sought thereby. Bothpolyclonal antibodies and monoclonal antibodies have been reported asuseful in these strategies (Rowland et al., (1986) Cancer Immunol.Immunother., 21:183-87). Drugs used in these methods include daunomycin,doxorubicin, methotrexate, and vindesine (Rowland et al., (1986) supra).Toxins used in antibody-toxin conjugates include bacterial toxins suchas diphtheria toxin, plant toxins such as ricin, small molecule toxinssuch as geldanamycin (Mandler et al (2000) Jour. of the Nat. CancerInst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med. Chem.Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem.13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl.Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al (1998)Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342). Thetoxins may affect their cytotoxic and cytostatic effects by mechanismsincluding tubulin binding, DNA binding, or topoisomerase inhibition.Some cytotoxic drugs tend to be inactive or less active when conjugatedto large antibodies or protein receptor ligands.

Examples of antibody drug conjugates are, ZEVALIN® (ibritumomabtiuxetan, Biogen/Idec) which is an antibody-radioisotope conjugatecomposed of a murine IgG1 kappa monoclonal antibody directed against theCD20 antigen found on the surface of normal and malignant B lymphocytesand ¹¹¹In or ⁹⁰Y radioisotope bound by a thiourea linker-chelator(Wiseman et al (2000) Eur. Jour. Nucl. Med. 27(7):766-77; Wiseman et al(2002) Blood 99(12):4336-42; Witzig et al (2002) J. Clin. Oncol.20(10):2453-63; Witzig et al (2002) J. Clin. Oncol. 20(15):3262-69).

Additionally, MYLOTARG™ (gemtuzumab ozogamicin, Wyeth Pharmaceuticals),an antibody drug conjugate composed of a human CD33 antibody linked tocalicheamicin, was approved in 2000 for the treatment of acute myeloidleukemia by injection (Drugs of the Future (2000) 25(7):686; U.S. Pat.Nos. 4,970,198; 5,079,233; 5,585,089; 5,606,040; 5,693,762; 5,739,116;5,767,285; 5,773,001).

In addition, Cantuzumab mertansine (Immunogen, Inc.), an antibody drugconjugate composed of the huC242 antibody linked via the disulfidelinker SPP to the maytansinoid drug moiety, DM1, is advancing into PhaseII trials for the treatment of cancers that express CanAg, such ascolon, pancreatic, gastric, and others.

Additionally, MLN-2704 (Millennium Pharm., BZL Biologics, ImmunogenInc.), an antibody drug conjugate composed of the anti-prostate specificmembrane antigen (PSMA) monoclonal antibody linked to the maytansinoiddrug moiety, DM1, is under development for the potential treatment ofprostate tumors.

Finally, the auristatin peptides, such as monomethyl auristatin E(MMAE), synthetic analogs of dolastatin, were conjugated to chimericmonoclonal antibodies cBR96 (specific to Lewis Y on carcinomas) andcAC10 (specific to CD30 on hematological malignancies) (Doronina et al(2003) Nature Biotechnology 21(7):778-784). The cAC10 is undertherapeutic development.

Further, chemotherapeutic agents useful in the generation of ADCs aredescribed herein. Enzymatically active toxins and fragments thereof thatcan be used include diphtheria A chain, nonbinding active fragments ofdiphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricinA chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes. See, e.g., WO 93/21232 published Oct.28, 1993. A variety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re. Conjugates of the antibody and cytotoxic agent are made usinga variety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate),aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al (1987) Science, 238:1098.Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody (WO94/11026).

Conjugates of an antibody and one or more small molecule toxins, such asa calicheamicin, maytansinoids, dolastatins, auristatins, atrichothecene, and CC1065, and the derivatives of these toxins that havetoxin activity, are also contemplated herein.

III(A). Maytansinoids Maytansine compounds suitable for use asmaytansinoid drug moieties are well known in the art, and can beisolated from natural sources according to known methods, produced usinggenetic engineering techniques (see Yu et al (2002) PNAS 99:7968-7973),or maytansinol and maytansinol analogues prepared syntheticallyaccording to known methods.

Exemplary maytansinoid drug moieties include those having a modifiedaromatic ring, such as: C-19-dechloro (U.S. Pat. No. 4,256,746)(prepared by lithium aluminum hydride reduction of ansamytocin P2);C-20-hydroxy (or C-20-demethyl)+/−C-19-dechloro (U.S. Pat. Nos.4,361,650 and 4,307,016) (prepared by demethylation using Streptomycesor Actinomyces or dechlorination using LAH); and C-20-demethoxy,C-20-acyloxy (—OCOR), +/−dechloro (U.S. Pat. No. 4,294,757) (prepared byacylation using acyl chlorides). and those having modifications at otherpositions

Exemplary maytansinoid drug moieties also include those havingmodifications such as: C-9-SH (U.S. Pat. No. 4,424,219) (prepared by thereaction of maytansinol with H₂S or P₂S₅);C-14-alkoxymethyl(demethoxy/CH₂OR)(U.S. Pat. No. 4,331,598);C-14-hydroxymethyl or acyloxymethyl (CH₂OH or CH₂OAc) (U.S. Pat. No.4,450,254) (prepared from Nocardia); C-15-hydroxy/acyloxy (U.S. Pat. No.4,364,866) (prepared by the conversion of maytansinol by Streptomyces);C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated fromTrewia nudlflora); C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and4,322,348) (prepared by the demethylation of maytansinol byStreptomyces); and 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by thetitanium trichloride/LAH reduction of maytansinol).

ADCs containing maytansinoids, methods of making same, and theirtherapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020;5,416,064; 6,441,163 and European Patent EP 0 425 235 B1, thedisclosures of which are hereby expressly incorporated by reference. Liuet al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) described ADCscomprising a maytansinoid designated DM1 linked to the monoclonalantibody C242 directed against human colorectal cancer. The conjugatewas found to be highly cytotoxic towards cultured colon cancer cells,and showed antitumor activity in an in vivo tumor growth assay. Chari etal., Cancer Research 52:127-131 (1992) describe ADCs in which amaytansinoid was conjugated via a disulfide linker to the murineantibody A7 binding to an antigen on human colon cancer cell lines, orto another murine monoclonal antibody TA. 1 that binds the HER-2/neuoncogene. The cytotoxicity of the TA.1-maytansonoid conjugate was testedin vitro on the human breast cancer cell line SK-BR-3, which expresses3×10⁵ HER-2 surface antigens per cell. The drug conjugate achieved adegree of cytotoxicity similar to the free maytansinoid drug, whichcould be increased by increasing the number of maytansinoid moleculesper antibody molecule. The A7-maytansinoid conjugate showed low systemiccytotoxicity in mice.

III(B). Auristatins and Dolastatins

In some embodiments, the ADC comprises an antibody of the inventionconjugated to dolastatins or dolostatin peptidic analogs andderivatives, the auristatins (U.S. Pat. Nos. 5,635,483; 5,780,588).Dolastatins and auristatins have been shown to interfere withmicrotubule dynamics, GTP hydrolysis, and nuclear and cellular division(Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584)and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity(Pettit et al (1998) Antimicrob. Agents Chemother. 42:2961-2965). Thedolastatin or auristatin drug moiety may be attached to the antibodythrough the N (amino) terminus or the C (carboxyl) terminus of thepeptidic drug moiety (WO 02/088172).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties DE and DF, disclosed in “Senter etal, Proceedings of the American Association for Cancer Research, Volume45, Abstract Number 623, presented Mar. 28, 2004 and described in UnitedStates Patent Publication No. 2005/0238649, the disclosure of which isexpressly incorporated by reference in its entirety.

An exemplary auristatin embodiment is MMAE (wherein the wavy lineindicates the covalent attachment to a linker (L) of an antibody drugconjugate).

Another exemplary auristatin embodiment is MMAF, wherein the wavy lineindicates the covalent attachment to a linker (L) of an antibody drugconjugate (US 2005/0238649):

Additional exemplary embodiments comprising MMAE or MMAF and variouslinker components (described further herein) have the followingstructures and abbreviations (wherein Ab means antibody and p is 1 toabout 8):

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to the liquidphase synthesis method (see E. Schrader and K. Libke, “The Peptides”,volume 1, pp 76-136, 1965, Academic Press) that is well known in thefield of peptide chemistry. The auristatin/dolastatin drug moieties maybe prepared according to the methods of: U.S. Pat. Nos. 5,635,483;5,780,588; Pettit et al (1989) J. Am. Chem. Soc. 111:5463-5465; Pettitet al (1998) Anti-Cancer Drug Design 13:243-277; Pettit, G. R., et al.Synthesis, 1996, 719-725; Pettit et al (1996) J. Chem. Soc. PerkinTrans. 1 5:859-863; and Doronina (2003) Nat Biotechnol 21(7):778-784.

III(C). Calicheamicin

In other embodiments, the ADC comprises an antibody of the inventionconjugated to one or more calicheamicin molecules. The calicheamicinfamily of antibiotics are capable of producing double-stranded DNAbreaks at sub-picomolar concentrations. For the preparation ofconjugates of the calicheamicin family, see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and5,877,296 (all to American Cyanamid Company). Structural analogues ofcalicheamicin which may be used include, but are not limited to, γ₁ ¹,α₂ ¹, α₃ ^(I), N-acetyl-γ₁ ^(I), PSAG and θ₁ ^(I) (Hinman et al., CancerResearch 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928(1998) and the aforementioned U.S. patents to American Cyanamid).Another anti-tumor drug that the antibody can be conjugated is QFA whichis an antifolate. Both calicheamicin and QFA have intracellular sites ofaction and do not readily cross the plasma membrane. Therefore, cellularuptake of these agents through antibody mediated internalization greatlyenhances their cytotoxic effects.

III(D). Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the antibodies of theinvention include BCNU, streptozoicin, vincristine and 5-fluorouracil,the family of agents known collectively LL-E33288 complex described inU.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat.No. 5,877,296).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an ADC formed between anantibody and a compound with nucleolytic activity (e.g., a ribonucleaseor a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated antibodies. Examples includeAt²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² andradioactive isotopes of Lu. When the conjugate is used for detection, itmay comprise a radioactive atom for scintigraphic studies, for exampletc^(99m) or I¹²³, or a spin label for nuclear magnetic resonance (NMR)imaging (also known as magnetic resonance imaging, mri), such asiodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13,nitrogen-15, oxygen-17, gadolinium, manganese or iron.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as tc^(99m) or I¹²³, Re¹⁸⁶, Re¹⁸⁸ and In¹¹¹ can be attachedvia a cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Frakcr et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

IV.) Antibody-Drug Conjugate Compounds which Bind 158P1D7

The present invention provides, inter alia, antibody-drug conjugatecompounds for targeted delivery of drugs. The inventors have made thediscovery that the antibody-drug conjugate compounds have potentcytotoxic and/or cytostatic activity against cells expressing 158P1D7.The antibody-drug conjugate compounds comprise an Antibody unitcovalently linked to at least one Drug unit. The Drug units can becovalently linked directly or via a Linker unit (-LU-).

In some embodiments, the antibody drug conjugate compound has thefollowing formula:

L-(LU-D)_(p)  (I)

or a pharmaceutically acceptable salt or solvate thereof; wherein:

-   -   L is the Antibody unit, e.g., 158P1D7 MAb of the present        invention, and    -   (LU-D) is a Linker unit-Drug unit moiety, wherein:    -   LU- is a Linker unit, and    -   -D is a drug unit having cytostatic or cytotoxic activity        against a target cell; and    -   p is an integer from 1 to 20.

In some embodiments, p ranges from 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p ranges from2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4 or 2 to 3. Inother embodiments, p is 1, 2, 3, 4, 5 or 6. In some embodiments, p is 2or 4.

In some embodiments, the antibody drug conjugate compound has thefollowing formula:

L-(A_(a)-W_(w)—Y_(y)-D)_(p)  (II)

or a pharmaceutically acceptable salt or solvate thereof, wherein:

-   -   L is the Antibody unit, e.g., 158P1D7 MAb; and    -   -A_(a)-W_(w)—Y_(y)— is a Linker unit (LU), wherein:    -   -A- is a Stretcher unit,    -   a is 0 or 1,    -   each —W— is independently an Amino Acid unit,    -   w is an integer ranging from 0 to 12,    -   —Y— is a self-immolative spacer unit,    -   y is 0, 1 or 2;    -   -D is a drug units having cytostatic or cytotoxic activity        against the target cell; and    -   p is an integer from 1 to 20.

In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0, 1 or 2. Insome embodiments, a is 0 or 1, w is 0 or 1, and y is 0 or 1. In someembodiments, p ranges from 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p ranges from 2 to 8,2 to 7, 2 to 6, 2 to 5, 2 to 4 or 2 to 3. In other embodiments, p is 1,2, 3, 4, 5 or 6. In some embodiments, p is 2 or 4. In some embodiments,when w is not zero, y is 1 or 2. In some embodiments, when w is 1 to 12,y is 1 or 2. In some embodiments, w is 2 to 12 and y is 1 or 2. In someembodiments, a is 1 and w and y are 0.

For compositions comprising a plurality antibodies, the drug loading isrepresented by p, the average number of drug molecules per Antibody.Drug loading may range from 1 to 20 drugs (D) per Antibody. The averagenumber of drugs per antibody in preparation of conjugation reactions maybe characterized by conventional means such as mass spectroscopy, ELISAassay, and HPLC. The quantitative distribution ofAntibody-Drug-Conjugates in terms of p may also be determined. In someinstances, separation, purification, and characterization of homogeneousAntibody-Drug-conjugates where p is a certain value fromAntibody-Drug-Conjugates with other drug loadings may be achieved bymeans such as reverse phase HPLC or electrophoresis. In exemplaryembodiments, p is from 2 to 8.

The generation of Antibody-drug conjugate compounds can be accomplishedby any technique known to the skilled artisan. Briefly, theAntibody-drug conjugate compounds comprise 158P1D7 MAb as the Antibodyunit, a drug, and optionally a linker that joins the drug and thebinding agent. In a preferred embodiment, the Antibody is 158P1D7 MAbcomprising heavy and light chain variable regions of an antibodydesignated Ha15-10ac12 described above. In more preferred embodiment,the Antibody is 158P1D7 MAb comprising heavy and light chain of anantibody designated Ha15-10ac12 described above. A number of differentreactions are available for covalent attachment of drugs and/or linkersto binding agents. This is often accomplished by reaction of the aminoacid residues of the binding agent, e.g., antibody molecule, includingthe amine groups of lysine, the free carboxylic acid groups of glutamicand aspartic acid, the sulfhydryl groups of cysteine and the variousmoieties of the aromatic amino acids. One of the most commonly usednon-specific methods of covalent attachment is the carbodiimide reactionto link a carboxy (or amino) group of a compound to amino (or carboxy)groups of the antibody. Additionally, bifunctional agents such asdialdehydes or imidoesters have been used to link the amino group of acompound to amino groups of an antibody molecule. Also available forattachment of drugs to binding agents is the Schiff base reaction. Thismethod involves the periodate oxidation of a drug that contains glycolor hydroxy groups, thus forming an aldehyde which is then reacted withthe binding agent. Attachment occurs via formation of a Schiff base withamino groups of the binding agent. Isothiocyanates can also be used ascoupling agents for covalently attaching drugs to binding agents. Othertechniques are known to the skilled artisan and within the scope of thepresent invention.

In certain embodiments, an intermediate, which is the precursor of thelinker, is reacted with the drug under appropriate conditions. Incertain embodiments, reactive groups are used on the drug and/or theintermediate. The product of the reaction between the drug and theintermediate, or the derivatized drug, is subsequently reacted with the158P1D7 MAb under appropriate conditions.

Each of the particular units of the Antibody-drug conjugate compounds isdescribed in more detail herein. The synthesis and structure ofexemplary Linker units, Stretcher units, Amino Acid units,self-immolative Spacer unit, and Drug units are also described in U.S.Patent Application Publication Nos. 2003-0083263, 2005-0238649 and2005-0009751, each if which is incorporated herein by reference in itsentirety and for all purposes.

V.) Linker Units

Typically, the antibody-drug conjugate compounds comprise a Linker unitbetween the drug unit and the antibody unit. In some embodiments, thelinker is cleavable under intracellular conditions, such that cleavageof the linker releases the drug unit from the antibody in theintracellular environment. In yet other embodiments, the linker unit isnot cleavable and the drug is released, for example, by antibodydegradation.

In some embodiments, the linker is cleavable by a cleaving agent that ispresent in the intracellular environment (e.g., within a lysosome orendosome or caveolea). The linker can be, e.g., a peptidyl linker thatis cleaved by an intracellular peptidase or protease enzyme, including,but not limited to, a lysosomal or endosomal protease. In someembodiments, the peptidyl linker is at least two amino acids long or atleast three amino acids long. Cleaving agents can include cathepsins Band D and plasmin, all of which are known to hydrolyze dipeptide drugderivatives resulting in the release of active drug inside target cells(see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123).Most typical are peptidyl linkers that are cleavable by enzymes that arepresent in 158P1D7-expressing cells. For example, a peptidyl linker thatis cleavable by the thiol-dependent protease cathepsin-B, which ishighly expressed in cancerous tissue, can be used (e.g., a Phe-Leu or aGly-Phe-Leu-Gly linker (SEQ ID NO: 9)). Other examples of such linkersare described, e.g., in U.S. Pat. No. 6,214,345, incorporated herein byreference in its entirety and for all purposes. In a specificembodiment, the peptidyl linker cleavable by an intracellular proteaseis a Val-Cit linker or a Phe-Lys linker (see, e.g., U.S. Pat. No.6,214,345, which describes the synthesis of doxorubicin with the val-citlinker). One advantage of using intracellular proteolytic release of thetherapeutic agent is that the agent is typically attenuated whenconjugated and the serum stabilities of the conjugates are typicallyhigh.

In other embodiments, the cleavable linker is pH-sensitive, i.e.,sensitive to hydrolysis at certain pH values. Typically, thepH-sensitive linker hydrolyzable under acidic conditions. For example,an acid-labile linker that is hydrolyzable in the lysosome (e.g., ahydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide,orthoester, acetal, ketal, or the like) can be used. (See, e.g., U.S.Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999,Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem.264:14653-14661.) Such linkers are relatively stable under neutral pHconditions, such as those in the blood, but are unstable at below pH 5.5or 5.0, the approximate pH of the lysosome. In certain embodiments, thehydrolyzable linker is a thioether linker (such as, e.g., a thioetherattached to the therapeutic agent via an acylhydrazone bond (see, e.g.,U.S. Pat. No. 5,622,929).

In yet other embodiments, the linker is cleavable under reducingconditions (e.g., a disulfide linker). A variety of disulfide linkersare known in the art, including, for example, those that can be formedusing SATA (N-succinimidyl-S-acetylthioacetate), SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene),SPDB and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res.47:5924-5931; Wawrzynczak et al., In Immunoconjugates: AntibodyConjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed.,Oxford U. Press, 1987. See also U.S. Pat. No. 4,880,935.)

In yet other specific embodiments, the linker is a malonate linker(Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyllinker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a3′-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

In yet other embodiments, the linker unit is not cleavable and the drugis released by antibody degradation. (See U.S. Publication No.2005/0238649 incorporated by reference herein in its entirety and forall purposes).

Typically, the linker is not substantially sensitive to theextracellular environment. As used herein, “not substantially sensitiveto the extracellular environment,” in the context of a linker, meansthat no more than about 20%, typically no more than about 15%, moretypically no more than about 10%, and even more typically no more thanabout 5%, no more than about 3%, or no more than about 1% of thelinkers, in a sample of antibody-drug conjugate compound, are cleavedwhen the antibody-drug conjugate compound presents in an extracellularenvironment (e.g., in plasma). Whether a linker is not substantiallysensitive to the extracellular environment can be determined, forexample, by incubating with plasma the antibody-drug conjugate compoundfor a predetermined time period (e.g., 2, 4, 8, 16, or 24 hours) andthen quantitating the amount of free drug present in the plasma.

In other, non-mutually exclusive embodiments, the linker promotescellular internalization. In certain embodiments, the linker promotescellular internalization when conjugated to the therapeutic agent (i.e.,in the milieu of the linker-therapeutic agent moiety of theantibody-drug conjugate compound as described herein). In yet otherembodiments, the linker promotes cellular internalization whenconjugated to both the auristatin compound and the 158P1D7 MAb.

A variety of exemplary linkers that can be used with the presentcompositions and methods are described in WO 2004-010957, U.S.Publication No. 2006/0074008, U.S. Publication No. 20050238649, and U.S.Publication No. 2006/0024317 (each of which is incorporated by referenceherein in its entirety and for all purposes).

A “Linker unit” (LU) is a bifunctional compound that can be used to linka Drug unit and a Antibody unit to form an antibody-drug conjugatecompound. In some embodiments, the Linker unit has the formula:

-A_(a)-W_(w)—Y_(y)—

-   -   wherein:-A- is a Stretcher unit,    -   a is 0 or 1,    -   each —W— is independently an Amino Acid unit,    -   w is an integer ranging from 0 to 12,    -   —Y— is a self-immolative Spacer unit, and    -   y is 0, 1 or 2.

In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0, 1 or 2. Insome embodiments, a is 0 or 1, w is 0 or 1, and y is 0 or 1. In someembodiments, when w is 1 to 12, y is 1 or 2. In some embodiments, w is 2to 12 and y is 1 or 2. In some embodiments, a is 1 and w and y are 0.

VI.) The Stretcher Unit

The Stretcher unit (A), when present, is capable of linking an Antibodyunit to an Amino Acid unit (—W—), if present, to a Spacer unit (—Y—), ifpresent; or to a Drug unit (-D). Useful functional groups that can bepresent on a 158P1D7 MAb (e.g. Ha15-10ac12), either naturally or viachemical manipulation include, but are not limited to, sulfhydryl,amino, hydroxyl, the anomeric hydroxyl group of a carbohydrate, andcarboxyl. Suitable functional groups are sulfhydryl and amino. In oneexample, sulfhydryl groups can be generated by reduction of theintramolecular disulfide bonds of a 158P1D7 MAb. In another embodiment,sulfhydryl groups can be generated by reaction of an amino group of alysine moiety of a 158P1D7 MAb with 2-iminothiolane (Traut's reagent) orother sulfhydryl generating reagents. In certain embodiments, the158P1D7 MAb is a recombinant antibody and is engineered to carry one ormore lysines. In certain other embodiments, the recombinant 158P1D7 MAbis engineered to carry additional sulfhydryl groups, e.g., additionalcysteines.

In one embodiment, the Stretcher unit forms a bond with a sulfur atom ofthe Antibody unit. The sulfur atom can be derived from a sulfhydrylgroup of an antibody. Representative Stretcher units of this embodimentare depicted within the square brackets of Formulas IIIa and IIIb,wherein L-, —W—, —Y—, -D, w and y are as defined above, and R¹⁷ isselected from —C₁-C₁₀ alkylene-, —C₁-C₁₀ alkenylene-, —C₁-C₁₀alkynylene-, carbocyclo-, —O—(C₁-C₈ alkylene)-, O—(C₁-C₈ alkenylene)-,—O—(C₁-C₈ alkynylene)-, -arylene-, —C₁-C₁₀ alkylene-arylene-, —C₂-C₁₀alkenylene-arylene, —C₂-C₁₀ alkynylene-arylene, -arylene-C₁-C₁₀alkylene-, -arylene-C₂-C₁₀ alkenylene-, -arylene-C₂-C₁₀ alkynylene-,—C₁-C₁₀ alkylene-(carbocyclo)-, —C₂-C₁₀ alkenylene-(carbocyclo)-,—C₂-C₁₀ alkynylene-(carbocyclo)-, -(carbocyclo)-C₁-C₁₀ alkylene-,-(carbocyclo)-C₂-C₁₀ alkenylene-, -(carbocyclo)-C₂-C₁₀ alkynylene,-heterocyclo-, —C₁-C₁₀ alkylene-(heterocyclo)-, —C₂-C₁₀alkenylene-(heterocyclo)-, —C₂-C₁₀ alkynylene-(heterocyclo)-,-(heterocyclo)-C₁-C₁₀ alkylene-, -(heterocyclo)-C₂-C₁₀ alkenylene-,-(heterocyclo)-C₁-C₁₀ alkynylene-, —(CH₂CH₂O)_(r)—, or—(CH₂CH₂O)_(r)—CH₂—, and r is an integer ranging from 1-10, wherein saidalkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynyklene, aryl,carbocycle, carbocyclo, heterocyclo, and arylene radicals, whether aloneor as part of another group, are optionally substituted. In someembodiments, said alkyl, alkenyl, alkynyl, alkylene, alkenylene,alkynyklene, aryl, carbocyle, carbocyclo, heterocyclo, and aryleneradicals, whether alone or as part of another group, are unsubstituted.In some embodiments, R¹⁷ is selected from —C₁-C₁₀ alkylene-,-carbocyclo-, —O—(C₁-C₈ alkylene)-, -arylene-, —C₁-C₁₀alkylene-arylene-, -arylene-C₁-C₁₀ alkylene-, —C₁-C₁₀alkylene-(carbocyclo)-, -(carbocyclo)-C₁-C₁₀ alkylene-, —C₃-C₅heterocyclo-, —C₁-C₁₀ alkylene-(heterocyclo)-, -(heterocyclo)-C₁-C₁₀alkylene-, —(CH₂CH₂O)_(r)—, and —(CH₂CH₂O)_(r)—CH₂—; and r is an integerranging from 1-10, wherein said alkylene groups are unsubstituted andthe remainder of the groups are optionally substituted.

It is to be understood from all the exemplary embodiments that evenwhere not denoted expressly, from 1 to 20 drug moieties can be linked toan Antibody (p=1-20).

An illustrative Stretcher unit is that of Formula IIIa wherein R¹⁷ is—(CH₂)₅—:

Another illustrative Stretcher unit is that of Formula IIIa wherein R¹⁷is —(CH₂CH₂O)_(r)—CH₂—; and r is 2:

An illustrative Stretcher unit is that of Formula IIIa wherein R¹⁷ is-arylene- or arylene-C₁-C₁₀ alkylene-. In some embodiments, the arylgroup is an unsubstituted phenyl group.

Still another illustrative Stretcher unit is that of Formula IIIbwherein R⁷ is —(CH₂)₅—:

In certain embodiments, the Stretcher unit is linked to the Antibodyunit via a disulfide bond between a sulfur atom of the Antibody unit anda sulfur atom of the Stretcher unit. A representative Stretcher unit ofthis embodiment is depicted within the square brackets of Formula IV,wherein R¹⁷, L-, —W—, —Y—, -D, w and y are as defined above.

It should be noted that throughout this application, the S moiety in theformula below refers to a sulfur atom of the Antibody unit, unlessotherwise indicated by context.

In yet other embodiments, the Stretcher contains a reactive site thatcan form a bond with a primary or secondary amino group of an Antibody.Examples of these reactive sites include, but are not limited to,activated esters such as succinimide esters, 4 nitrophenyl esters,pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acidchlorides, sulfonyl chlorides, isocyanates and isothiocyanates.Representative Stretcher units of this embodiment are depicted withinthe square brackets of Formulas Va and Vb, wherein —R¹⁷—, L-, —W—, —Y—,-D, w and y are as defined above;

In some embodiments, the Stretcher contains a reactive site that isreactive to a modified carbohydrate's (—CHO) group that can be presenton an Antibody. For example, a carbohydrate can be mildly oxidized usinga reagent such as sodium periodate and the resulting (—CHO) unit of theoxidized carbohydrate can be condensed with a Stretcher that contains afunctionality such as a hydrazide, an oxime, a primary or secondaryamine, a hydrazine, a thiosemicarbazone, a hydrazine carboxylate, and anarylhydrazide such as those described by Kaneko et al., 1991,Bioconjugate Chem. 2:133-41. Representative Stretcher units of thisembodiment are depicted within the square brackets of Formulas VIa, VIb,and VIc, wherein —R¹⁷—, L-, —W—, —Y—, -D, w and y are as defined asabove.

VII.) The Amino Acid Unit

The Amino Acid unit (—W—), when present, links the Stretcher unit to theSpacer unit if the Spacer unit is present, links the Stretcher unit tothe Drug moiety if the Spacer unit is absent, and links the Antibodyunit to the Drug unit if the Stretcher unit and Spacer unit are absent.

W_(w)— can be, for example, a monopeptide, dipeptide, tripeptide,tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide,nonapeptide, decapeptide, undecapeptide or dodecapeptide unit. Each —W—unit independently has the formula denoted below in the square brackets,and w is an integer ranging from 0 to 12:

wherein R¹⁹ is hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl,p-hydroxybenzyl, —CH₂OH, —CH(OH)CH₃, —CH₂CH₂SCH₃, —CH₂CONH₂, —CH₂COOH,—CH₂CH₂CONH₂, —CH₂CH₂COOH, —(CH₂)₃NHC(═NH)NH₂, —(CH₂)₃NH₂,—(CH₂)₃NHCOCH₃, —(CH₂)₃NHCHO, —(CH₂)₄NHC(═NH)NH₂, —(CH₂)₄NH₂,—(CH₂)₄NHCOCH₃, —(CH₂)₄NHCHO, —(CH₂)₃NHCONH₂, —(CH₂)₄NHCONH₂,—CH₂CH₂CH(OH)CH₂NH₂, 2-pyridylmethyl-, 3-pyridylmethyl-,4-pyridylmethyl-, phenyl, cyclohexyl,

In some embodiments, the Amino Acid unit can be enzymatically cleaved byone or more enzymes, including a cancer or tumor-associated protease, toliberate the Drug unit (-D), which in one embodiment is protonated invivo upon release to provide a Drug (D).

In certain embodiments, the Amino Acid unit can comprise natural aminoacids. In other embodiments, the Amino Acid unit can comprisenon-natural amino acids. Illustrative Ww units are represented byformulas (VII)-(IX):

wherein R²⁰ and R²¹ are as follows:

R²⁰ R²¹ Benzyl (CH₂)₄NH₂; methyl (CH₂)₄NH₂; isopropyl (CH₂)₄NH₂;isopropyl (CH₂)₃NHCONH₂; benzyl (CH₂)₃NHCONH₂; isobutyl (CH₂)₃NHCONH₂;sec-butyl (CH₂)₃NHCONH₂;

(CH₂)₃NHCONH₂; benzyl methyl; benzyl (CH₂)₃NHC(═NH)NH₂;

wherein R²⁰, R²¹ and R²² are as follows:

R²⁰ R²¹ R²² benzyl benzyl (CH₂)₄NH₂; isopropyl benzyl (CH₂)₄NH₂; and Hbenzyl (CH₂)₄NH₂;

wherein R²⁰, R²¹, R²² and R²³ are as follows:

R²⁰ R²¹ R²² R²³ H benzyl isobutyl H; and methyl isobutyl methylisobutyl.

Exemplary Amino Acid units include, but are not limited to, units offormula VII where: R²⁰ is benzyl and R²¹ is —(CH₂)₄NH₂; R²⁰ is isopropyland R²¹ is —(CH₂)₄NH₂; or R²⁰ is isopropyl and R² is —(CH₂)₃NHCONH₂.Another exemplary Amino Acid unit is a unit of formula VIII wherein R²⁰is benzyl, R²¹ is benzyl, and R²² is —(CH₂)₄NH₂.

Useful —W_(w)— units can be designed and optimized in their selectivityfor enzymatic cleavage by a particular enzyme, for example, atumor-associated protease. In one embodiment, a —W_(w)— unit is thatwhose cleavage is catalyzed by cathepsin B, C and D, or a plasminprotease.

In one embodiment, —W_(w)— is a dipeptide, tripeptide, tetrapeptide orpentapeptide. When R¹⁹, R²⁰, R²¹, R²² or R²³ is other than hydrogen, thecarbon atom to which R¹⁹, R²⁰, R²¹, R²² or R²³ is attached is chiral.

Each carbon atom to which R¹⁹, R²⁰, R²¹, R²² or R²³ is attached isindependently in the (S) or (R) configuration.

In one aspect of the Amino Acid unit, the Amino Acid unit isvaline-citrulline (vc or val-cit). In another aspect, the Amino Acidunit is phenylalanine-lysine (i.e., fk). In yet another aspect of theAmino Acid unit, the Amino Acid unit is N-methylvaline-citrulline. Inyet another aspect, the Amino Acid unit is 5-aminovaleric acid, homophenylalanine lysine, tetraisoquinolinecarboxylate lysine,cyclohexylalanine lysine, isonepecotic acid lysine, beta-alanine lysine,glycine serine valine glutamine and isonepecotic acid.

VIII.) The Spacer Unit

The Spacer unit (—Y—), when present, links an Amino Acid unit to theDrug unit when an Amino Acid unit is present. Alternately, the Spacerunit links the Stretcher unit to the Drug unit when the Amino Acid unitis absent. The Spacer unit also links the Drug unit to the Antibody unitwhen both the Amino Acid unit and Stretcher unit are absent.

Spacer units are of two general types: non self-immolative orself-immolative. A non self-immolative Spacer unit is one in which partor all of the Spacer unit remains bound to the Drug moiety aftercleavage, particularly enzymatic, of an Amino Acid unit from theantibody-drug conjugate. Examples of a non self-immolative Spacer unitinclude, but are not limited to a (glycine-glycine) Spacer unit and aglycine Spacer unit (both depicted in Scheme 1) (infra). When aconjugate containing a glycine-glycine Spacer unit or a glycine Spacerunit undergoes enzymatic cleavage via an enzyme (e.g., a tumor-cellassociated-protease, a cancer-cell-associated protease or alymphocyte-associated protease), a glycine-glycine-Drug moiety or aglycine-Drug moiety is cleaved from L-Aa-Ww-. In one embodiment, anindependent hydrolysis reaction takes place within the target cell,cleaving the glycine-Drug moiety bond and liberating the Drug.

In some embodiments, a non self-immolative Spacer unit (—Y—) is -Gly-.In some embodiments, a non self-immolative Spacer unit (—Y—) is-Gly-Gly-.

In one embodiment, a Drug-Linker conjugate is provided in which theSpacer unit is absent (y=0), or a pharmaceutically acceptable salt orsolvate thereof.

Alternatively, a conjugate containing a self-immolative Spacer unit canrelease -D. As used herein, the term “self-immolative Spacer” refers toa bifunctional chemical moiety that is capable of covalently linkingtogether two spaced chemical moieties into a stable tripartite molecule.It will spontaneously separate from the second chemical moiety if itsbond to the first moiety is cleaved.

In some embodiments, —Y_(y)— is a p-aminobenzyl alcohol (PAB) unit (seeSchemes 2 and 3) whose phenylene portion is substituted with Q_(m)wherein Q is —C₁-C₈ alkyl, —C₁-C₈ alkenyl, —C₁-C₈ alkynyl, —O—(C₁-C₈alkyl), —O—(C₁-C₈ alkenyl), —O—(C₁-C₈ alkynyl), -halogen, -nitro or-cyano; and m is an integer ranging from 0-4. The alkyl, alkenyl andalkynyl groups, whether alone or as part of another group, can beoptionally substituted.

In some embodiments, —Y— is a PAB group that is linked to —W_(w)— viathe amino nitrogen atom of the PAB group, and connected directly to -Dvia a carbonate, carbamate or ether group. Without being bound by anyparticular theory or mechanism, Scheme 2 depicts a possible mechanism ofDrug release of a PAB group which is attached directly to -D via acarbamate or carbonate group as described by Toki et al., 2002, J. Org.Chem. 67:1866-1872.

In Scheme 2, Q is —C₁-C₈ alkyl, —C₁-C₈ alkenyl, —C₁-C₈ alkynyl,—O—(C₁-C₈ alkyl), —O—(C₁-C₈ alkenyl), —O—(C₁-C₈ alkynyl), -halogen,-nitro or -cyano; m is an integer ranging from 0-4; and p ranges from 1to about 20. The alkyl, alkenyl and alkynyl groups, whether alone or aspart of another group, can be optionally substituted.

Without being bound by any particular theory or mechanism, Scheme 3depicts a possible mechanism of Drug release of a PAB group which isattached directly to -D via an ether or amine linkage, wherein Dincludes the oxygen or nitrogen group that is part of the Drug unit.

In Scheme 3, Q is —C₁-C₈ alkyl, —C₁-C₈ alkenyl, —C₁-C₈ alkynyl,—O—(C₁-C₈ alkyl), —O—(C₁-C₈ alkenyl), —O—(C₁-C₈ alkynyl), -halogen,-nitro or -cyano; m is an integer ranging from 0-4; and p ranges from 1to about 20. The alkyl, alkenyl and alkynyl groups, whether alone or aspart of another group, can be optionally substituted.

Other examples of self-immolative spacers include, but are not limitedto, aromatic compounds that are electronically similar to the PAB groupsuch as 2-aminoimidazol-5-methanol derivatives (Hay et al., 1999,Bioorg. Med. Chem. Lett. 9:2237) and ortho or para-aminobenzylacetals.Spacers can be used that undergo cyclization upon amide bond hydrolysis,such as substituted and unsubstituted 4-aminobutyric acid amides(Rodrigues et al., 1995, Chemistry Biology 2:223), appropriatelysubstituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm etal., 1972, J. Amer. Chem. Soc. 94:5815) and 2-aminophenylpropionic acidamides (Amsberry et al., 1990, J. Org. Chem. 55:5867). Elimination ofamine-containing drugs that are substituted at the α-position of glycine(Kingsbury et al., 1984, J. Med. Chem. 27:1447) are also examples ofself-immolative spacers.

In one embodiment, the Spacer unit is a branchedbis(hydroxymethyl)-styrene (BHMS) unit as depicted in Scheme 4, whichcan be used to incorporate and release multiple drugs.

In Scheme 4, Q is —C₁-C₈ alkyl, —C₁-C₈ alkenyl, —C₁-C₈ alkynyl,—O—(C₁-C₈ alkyl), —O—(C₁-C₈ alkenyl), —O—(C₁-C₈ alkynyl), -halogen,-nitro or -cyano; m is an integer ranging from 0-4; n is 0 or 1; and pranges raging from 1 to about 20. The alkyl, alkenyl and alkynyl groups,whether alone or as part of another group, can be optionallysubstituted.

In some embodiments, the -D moieties are the same. In yet anotherembodiment, the -D moieties are different.

In one aspect, Spacer units (—Y_(y)—) are represented by Formulas(X)-(XII):

wherein Q is —C₁-C₈ alkyl, —C₁-C₈ alkenyl, —C₁-C₈ alkynyl, —O—(C₁-C₈alkyl), —O—(C₁-C₈ alkenyl), —O—(C₁-C₈ alkynyl), -halogen, -nitro or-cyano; and m is an integer ranging from 0-4. The alkyl, alkenyl andalkynyl groups, whether alone or as part of another group, can beoptionally substituted.

Embodiments of the Formula I and II comprising antibody-drug conjugatecompounds can include:

wherein w and y are each 0, 1 or 2, and,

wherein w and y are each 0,

IX.) The Drug Unit

The Drug moiety (D) can be any cytotoxic, cytostatic or immunomodulatory(e.g., immunosuppressive) or drug. D is a Drug unit (moiety) having anatom that can form a bond with the Spacer unit, with the Amino Acidunit, with the Stretcher unit or with the Antibody unit. In someembodiments, the Drug unit D has a nitrogen atom that can form a bondwith the Spacer unit. As used herein, the terms “Drug unit” and “Drugmoiety” are synonymous and used interchangeably.

Useful classes of cytotoxic or immunomodulatory agents include, forexample, antitubulin agents, DNA minor groove binders, DNA replicationinhibitors, and alkylating agents.

In some embodiments, the Drug is an auristatin, such as auristatin E(also known in the art as a derivative of dolastatin-10) or a derivativethereof. The auristatin can be, for example, an ester formed betweenauristatin E and a keto acid. For example, auristatin E can be reactedwith paraacetyl benzoic acid or benzoylvaleric acid to produce AEB andAEVB, respectively. Other typical auristatins include AFP, MMAF, andMMAE. The synthesis and structure of exemplary auristatins are describedin U.S. Patent Application Publication Nos. 2003-0083263, 2005-0238649and 2005-0009751; International Patent Publication No. WO 04/010957,International Patent Publication No. WO 02/088172, and U.S. Pat. Nos.6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149;5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284; 5,504,191;5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278;4,816,444; and 4,486,414, each of which is incorporated by referenceherein in its entirety and for all purposes.

Auristatins have been shown to interfere with microtubule dynamics andnuclear and cellular division and have anticancer activity. Auristatinsbind tubulin and can exert a cytotoxic or cytostatic effect on a158P1D7-expressing cell. There are a number of different assays, knownin the art, which can be used for determining whether an auristatin orresultant antibody-drug conjugate exerts a cytostatic or cytotoxiceffect on a desired cell line.

Methods for determining whether a compound binds tubulin are known inthe art. See, for example, Muller et al., Anal. Chem 2006, 78,4390-4397; Hamel et al., Molecular Pharmacology, 1995 47: 965-976; andHamel et al., The Journal of Biological Chemistry, 1990 265:28,17141-17149. For purposes of the present invention, the relativeaffinity of a compound to tubulin can be determined. Some preferredauristatins of the present invention bind tubulin with an affinityranging from 10 fold lower (weaker affinity) than the binding affinityof MMAE to tubulin to 10 fold, 20 fold or even 100 fold higher (higheraffinity) than the binding affinity of MMAE to tubulin.

In some embodiments, -D is an auristatin of the formula D_(E) or D_(F):

or a pharmaceutically acceptable salt or solvate form thereof;wherein, independently at each location:

the wavy line indicates a bond;

R² is —C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl, or —C₂-C₂₀ alkynyl;

R³ is —H, —C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl, —C₂-C₂₀ alkynyl, -carbocycle,—C₁-C₂₀ alkylene (carbocycle), —C₂-C₂₀ alkenylene(carbocycle), —C₂-C₂₀alkynylene(carbocycle), -aryl, —C₁-C₂₀ alkylene(aryl), —C₂-C₂₀alkenylene(aryl), —C₂-C₂₀ alkynylene(aryl), heterocycle, —C₁-C₂₀alkylene(heterocycle), —C₂-C₂₀ alkenylene(heterocycle), or —C₂-C₂₀alkynylene(heterocycle);

R⁴ is —H, —C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl, —C₂-C₂₀ alkynyl, carbocycle,—C₁-C₂₀ alkylene (carbocycle), —C₂-C₂₀ alkenylene(carbocycle), —C₂-C₂₀alkynylene(carbocycle), aryl, —C₁-C₂₀ alkylene(aryl), —C₂-C₂₀alkenylene(aryl), —C₂-C₂₀ alkynylene(aryl), -heterocycle, —C₁-C₂₀alkylene(heterocycle), —C₂-C₂₀ alkenylene(heterocycle), or —C₂-C₂₀alkynylene(heterocycle);

R⁵ is —H or —C₁-C₈ alkyl;

or R⁴ and R⁵ jointly form a carbocyclic ring and have the formula—(CR^(a)R^(b))_(s)— wherein R^(a) and Rb are independently —H, —C₁-C₂₀alkyl, —C₂-C₂₀ alkenyl, —C₂-C₂₀ alkynyl, or -carbocycle and s is 2, 3,4, 5 or 6,

R⁶ is —H, —C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl, or —C₂-C₂₀ alkynyl;

R⁷ is —H, —C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl, —C₂-C₂₀ alkynyl, carbocycle,—C₁-C₂₀ alkylene (carbocycle), —C₂-C₂₀ alkenylene(carbocycle), —C₂-C₂₀alkynylene(carbocycle), -aryl, —C₁-C₂₀ alkylene(aryl), —C₂-C₂₀alkenylene(aryl), —C₂-C₂₀ alkynylene(aryl), heterocycle, —C₁-C₂₀alkylene(heterocycle), —C₂-C₂₀ alkenylene(heterocycle), or —C₂-C₂₀alkynylene(heterocycle);

each R⁸ is independently —H, —OH, —C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl,—C₂-C₂₀ alkynyl, —O—(C₁-C₂₀ alkyl), —O—(C₂-C₂₀ alkenyl), —O—(C₁-C₂₀alkynyl), or -carbocycle;

R⁹ is —H, —C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl, or —C₂-C₂₀ alkynyl;

R²⁴ is -aryl, -heterocycle, or -carbocycle;

R²⁵ is —H, C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl, —C₂-C₂₀ alkynyl, -carbocycle,—O—(C₁-C₂₀ alkyl), —O—(C₂-C₂₀ alkenyl), —O—(C₂-C₂₀ alkynyl), or ORwherein R¹⁸ is —H, a hydroxyl protecting group, or a direct bond whereOR¹⁸ represents ═O;

R²⁶ is —H, —C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl, or —C₂-C₂₀ alkynyl, -aryl,-heterocycle, or -carbocycle; R¹⁰ is -aryl or -heterocycle;

Z is —O, —S, —NH, or —NR¹², wherein R¹² is —C₁-C₂₀ alkyl, —C₂-C₂₀alkenyl, or —C₂-C₂₀ alkynyl;

R¹¹ is —H, —C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl, —C₂-C₂₀ alkynyl, -aryl,-heterocycle, —(R¹³O)_(m)—R¹⁴, or —(R¹³O)_(m)—CH(R¹⁵)₂;

m is an integer ranging from 1-1000;

R¹³ is —C₂-C₂₀ alkylene, —C₂-C₂₀ alkenylene, or —C₂-C₂₀ alkynylene;

R¹⁴ is —H, —C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl, or —C₂-C₂₀ alkynyl;

each occurrence of R¹⁵ is independently —H, —COOH, —(CH₂)_(n)—N(R¹⁶)₂,—(CH₂)_(n)—SO₃H, —(CH₂)_(n)—SO₃—C₁-C₂₀ alkyl, —(CH₂)_(n)—SO₃—C₂-C₂₀alkenyl, or —(CH₂)_(n)—SO₃—C₂-C₂₀ alkynyl;

each occurrence of R¹⁶ is independently —H, —C₁-C₂₀ alkyl, —C₂-C₂₀alkenyl, —C₂-C₂₀ alkynyl or —(CH₂)_(n)—COOH; and

n is an integer ranging from 0 to 6;

wherein said alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynyklene,aryl, carbocyle, and heterocycle radicals, whether alone or as part ofanother group, are optionally substituted.

Auristatins of the formula D_(E) include those wherein said alkyl,alkenyl, alkynyl, alkylene, alkenylene, alkynyklene, aryl, carbocyle,and heterocycle radicals are unsubstituted.

Auristatins of the formula D_(E) include those wherein the groups of R²,R³, R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ are unsubstituted and the groups of R¹⁹,R²⁰ and R²¹ are optionally substituted as described herein.

Auristatins of the formula D_(E) include those wherein:

R² is C₁-C₈ alkyl;

R³, R⁴ and R⁷ are independently selected from —H, —C₁-C₂₀ alkyl, —C₂-C₂₀alkenyl, —C₂-C₂₀ alkynyl, monocyclic C₃-C₆ carbocycle, —C₁-C₂₀alkylene(monocyclic C₃-C₆ carbocycle), —C₂-C₂₀ alkenylene(monocyclicC₃-C₆ carbocycle), —C₂-C₂₀ alkynylene(monocyclic C₃-C₆ carbocycle),C₆-C₁₀ aryl, —C₁-C₂₀ alkylene(C₆-C₁₀ aryl), —C₂-C₂₀ alkenylene(C₆-C₁₀aryl), —C₂-C₂₀ alkynylene(C₆-C₁₀ aryl), heterocycle, —C₁-C₂₀alkylene(heterocycle), —C₂-C₂₀ alkenylene(heterocycle), or —C₂-C₂₀alkynylene(heterocycle); wherein said alkyl, alkenyl, alkynyl, alkylene,alkenylene, alkynylene, carbocycle, aryl and heterocycle radicals areoptionally substituted;

R⁵ is —H;

R⁶ is —C₁-C₈ alkyl;

each R⁸ is independently selected from —OH, —O—(C₁-C₂₀ alkyl),—O—(C₂-C₂₀ alkenyl), or —O—(C₂-C₂₀ alkynyl) wherein said alkyl, alkenyl,and alkynyl radicals are optionally substituted;

R⁹ is —H or —C₁-C₈ alkyl;

R²⁴ is optionally substituted -phenyl;

R²⁵ is —OR¹⁸; wherein R¹⁸ is H, a hydroxyl protecting group, or a directbond where OR¹⁸ represents ═O;

R²⁶ is selected from —H, —C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl, —C₂-C₂₀alkynyl, or -carbocycle; wherein said alkyl, alkenyl, alkynyl andcarbocycle radicals are optionally substituted; or a pharmaceuticallyacceptable salt or solvate form thereof.

Auristatins of the formula D_(E) include those wherein:

R² is methyl;

R³ is —H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, or C₂-C₈ alkynyl, wherein saidalkyl, alkenyl and alkynyl radicals are optionally substituted;

R⁴ is —H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, monocyclic C₃-C₆carbocycle, —C₆-C₁₀ aryl, —C₁-C₈ alkylene(C₆-C₁₀ aryl), —C₂-C₈alkenylene(C₆-C₁₀ aryl), —C₂-C₈ alkynylene(C₆-C₁₀ aryl), —C₁-C₈ alkylene(monocyclic C₃-C₆ carbocycle), —C₂-C₈ alkenylene (monocyclic C₃-C₆carbocycle), —C₂-C₈ alkynylene(monocyclic C₃-C₆ carbocycle); whereinsaid alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl andcarbocycle radicals whether alone or as part of another group areoptionally substituted;

R⁵ is —H;

R⁶ is methyl;

R⁷ is —C₁-C₈ alkyl, —C₂-C₈ alkenyl or —C₂-C₈ alkynyl;

each R⁸ is methoxy;

R⁹ is —H or —C₁-C₈ alkyl;

R²⁴ is -phenyl;

R²⁵ is —OR¹⁸; wherein R¹⁸ is H, a hydroxyl protecting group, or a directbond where OR¹⁸ represents ═O;

R²⁶ is methyl;

or a pharmaceutically acceptable salt form thereof.

Auristatins of the formula D_(E) include those wherein: R² is methyl; R³is —H or —C₁-C₃ alkyl; R⁴ is —C₁-C₈ alkyl; R⁵ is —H; R⁶ is methyl; R⁷ isisopropyl or sec-butyl; R⁸ is methoxy; R⁹ is —H or —C₁-C₈ alkyl; R²⁴ isphenyl; R²⁵ is —OR¹⁸; wherein R¹⁸ is —H, a hydroxyl protecting group, ora direct bond where OR represents=O; and R²⁶ is methyl; or apharmaceutically acceptable salt or solvate form thereof.

Auristatins of the formula D_(E) include those wherein:

R² is methyl or C₁-C₃ alkyl,

R³ is —H or —C₁-C₃ alkyl;

R⁴ is —C₁-C₈ alkyl;

R⁵ is H;

R⁶ is C₁-C₃ alkyl;

R⁷ is —C₁-C₈ alkyl;

R⁸ is —C₁-C₃ alkoxy;

R⁹ is —H or —C₁-C₈ alkyl;

R²⁴ is phenyl;

R²⁵ is —OR¹⁸; wherein R¹⁸ is —H, a hydroxyl protecting group, or adirect bond where OR¹⁸ represents ═O; and

R²⁶ is —C₁-C₃ alkyl;

or a pharmaceutically acceptable salt form thereof.

Auristatins of the formula D_(F) include those wherein:

R² is methyl;

R³, R⁴, and R⁷ are independently selected from —H, —C₁-C₂₀ alkyl,—C₂-C₂₀ alkenyl, —C₂-C₂₀ alkynyl, monocyclic C₃-C₆ carbocycle, —C₁-C₂₀alkylene(monocyclic C₃-C₆ carbocycle), —C₂-C₂₀ alkenylene(monocyclicC₃-C₆ carbocycle), —C₂-C₂₀ alkynylene(monocyclic C₃-C₆ carbocycle),—C₆-C₁₀ aryl, —C₁-C₂₀ alkylene(C₆-C₁₀ aryl), —C₂-C₂₀ alkenylene(C₆-C₁₀aryl), —C₂-C₂₀ alkynylene(C₆-C₁₀ aryl), heterocycle, —C₁-C₂₀alkylene(heterocycle), —C₂-C₂₀ alkenylene(heterocycle), or —C₂-C₂₀alkynylene(heterocycle); wherein said alkyl, alkenyl, alkynyl, alkylene,alkenylene, alkynylene, carbocycle, aryl and heterocycle radicalswhether alone or as part of another group are optionally substituted;

R⁵ is —H;

R⁶ is methyl;

each R⁸ is methoxy;

R⁹ is —H, —C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl, or —C₂-C₂₀ alkynyl; whereinsaid alkyl, alkenyl and alkynyl radical are optionally substituted;

R¹⁰ is optionally substituted aryl or optionally substitutedheterocycle;

Z is —O—, —S—, —NH—, or —NR¹², wherein R¹² is —C₁-C₂₀ alkyl, —C₂-C₂₀alkenyl, or —C₂-C₂₀ alkynyl, each of which is optionally substituted;

R¹¹ is —H, —C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl, —C₂-C₂₀ alkynyl, -aryl,-heterocycle, —(R¹³O)_(m)—R¹⁴, or —(R¹³O)_(m)—CH(R¹⁵)₂, wherein saidalkyl, alkenyl, alkynyl, aryl and heterocycle radicals are optionallysubstituted;

m is an integer ranging from 1-1000 or m=0;

R¹³ is —C₂-C₂₀ alkylene, —C₂-C₂₀ alkenylene, or —C₂-C₂₀ alkynylene, eachof which is optionally substituted;

R¹⁴ is —H, —C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl, or —C₂-C₂₀ alkynyl whereinsaid alkyl, alkenyl and alkynyl radicals are optionally substituted;

each occurrence of R¹⁵ is independently —H, —COOH, —(CH₂)_(n)—N(R¹⁶)₂,—(CH₂)_(n)—SO₃H, —(CH₂)_(n)—SO₃—C₁-C₂₀ alkyl, —(CH₂)_(n)—SO₃—C₂-C₂₀alkenyl, or —(CH₂)_(n)—SO₃—C₂-C₂₀ alkynyl wherein said alkyl, alkenyland alkynyl radicals are optionally substituted;

each occurrence of R¹⁶ is independently —H, —C₁-C₂₀ alkyl, —C₂-C₂₀alkenyl, —C₂-C₂₀ alkynyl or —(CH₂)_(n)—COOH wherein said alkyl, alkenyland alkynyl radicals are optionally substituted;

n is an integer ranging from 0 to 6;

or a pharmaceutically acceptable salt thereof.

In certain of these embodiments, R¹⁰ is optionally substituted phenyl.

Auristatins of the formula D_(F) include those wherein the groups of R²,R³, R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ are unsubstituted and the groups of R¹⁰and R″ are as described herein.

Auristatins of the formula D_(F) include those wherein said alkyl,alkenyl, alkynyl, alkylene, alkenylene, alkynyklene, aryl, carbocyle,and heterocycle radicals are unsubstituted.

Auristatins of the formula D_(F) include those wherein:

R² is —C₁-C₃ alkyl; R³ is —H or —C₁-C₃ alkyl; R⁴ is —C₁-C₈ alkyl; R⁵ is—H; R⁶ is —C₁-C₃ alkyl; R⁷ is —C₁-C₈ alkyl; R⁸ is —C₁-C₃ alkoxy; R⁹ is—H or —C₁-C₈ alkyl; R¹⁰ is optionally substituted phenyl; Z is —O—, —S—,or —NH—; R¹ is as defined herein; or a pharmaceutically acceptable saltthereof.

Auristatins of the formula D_(F) include those wherein:

R² is methyl; R³ is —H or —C₁-C₃ alkyl; R⁴ is —C₁-C₈ alkyl; R⁵ is —H; R⁶is methyl; R⁷ is isopropyl or sec-butyl; R⁸ is methoxy; R⁹ is —H or—C₁-C₈ alkyl; R is optionally substituted phenyl; Z is —O—, —S—, or—NH—; and R¹¹ is as defined herein; or a pharmaceutically acceptablesalt thereof.

Auristatins of the formula D_(F) include those wherein:

R² is methyl; R³ is —H or —C₁-C₃ alkyl; R⁴ is —C₁-C₈ alkyl; R⁵ is —H; R⁶is methyl; R⁷ is isopropyl or sec-butyl; R⁸ is methoxy; R⁹ is —H orC₁-C₈ alkyl; R₁₀ is phenyl; and Z is —O— or —NH— and R¹¹ is as definedherein, preferably hydrogen; or a pharmaceutically acceptable salt formthereof.

Auristatins of the formula D_(F) include those wherein:

R² is —C₁-C₃ alkyl; R³ is —H or —C₁-C₃ alkyl; R⁴ is —C₁-C₈ alkyl; R⁵ is—H; R⁶ is —C₁-C₃ alkyl; R⁷ is —C₁-C₈ alkyl; R⁸ is —C₁-C₃ alkoxy; R⁹ is—H or —C₁-C₈ alkyl; R¹⁰ is phenyl; and Z is —O— or —NH— and R¹¹ is asdefined herein, preferably hydrogen; or a pharmaceutically acceptablesalt form thereof.

Auristatins of the formula D_(E) or D_(F) include those wherein R³, R⁴and R⁷ are independently isopropyl or sec-butyl and R⁵ is —H. In anexemplary embodiment, R³ and R⁴ are each isopropyl, R⁵ is H, and R⁷ issec-butyl. The remainder of the substituents are as defined herein.

Auristatins of the formula D_(E) or D_(F) include those wherein R² andR⁶ are each methyl, and R⁹ is H. The remainder of the substituents areas defined herein.

Auristatins of the formula D_(F) or D_(F) include those wherein eachoccurrence of R⁸ is —OCH₃. The remainder of the substituents are asdefined herein.

Auristatins of the formula D_(E) or D_(F) include those wherein R³ andR⁴ are each isopropyl, R² and R⁶ are each methyl, R⁵ is H, R⁷ issec-butyl, each occurrence of R⁸ is —OCH₃, and R⁹ is H. The remainder ofthe substituents are as defined herein.

Auristatins of the formula D_(F) include those wherein Z is —O— or —NH—.The remainder of the substituents are as defined herein.

Auristatins of the formula D_(F) include those wherein R¹⁰ is aryl. Theremainder of the substituents are as defined herein.

Auristatins of the formula D_(F) include those wherein R¹⁰ is -phenyl.The remainder of the substituents are as defined herein.

Auristatins of the formula D_(F) include those wherein Z is —O—, and R¹¹is H, methyl or t-butyl. The remainder of the substituents are asdefined herein.

Auristatins of the formula D_(F) include those wherein, when Z is —NH—,R¹ is —(R¹³O)_(m)—CH(R¹⁵)₂, wherein R⁵ is —(CH₂)_(n)—N(R¹⁶)₂, and R¹⁶ is—C₁-C₈ alkyl or —(CH₂)_(n)—COOH. The remainder of the substituents areas defined herein.

Auristatins of the formula D_(F) include those wherein when Z is —NH—,R¹¹ is —(R¹³O)_(m)—CH(R¹⁵)₂, wherein R¹⁵ is —(CH₂)_(n)—SO₃H. Theremainder of the substituents are as defined herein.

In preferred embodiments, when D is an auristatin of formula D_(E), w isan integer ranging from 1 to 12, preferably 2 to 12, y is 1 or 2, and ais preferably 1.

In some embodiments, wherein D is an auristatin of formula D_(F), a is 1and w and y are 0.

Illustrative Drug units (-D) include the drug units having the followingstructures:

or pharmaceutically acceptable salts or solvates thereof.

In one aspect, hydrophilic groups, such as but not limited totriethylene glycol esters (TEG) can be attached to the Drug Unit at R¹¹.Without being bound by theory, the hydrophilic groups assist in theinternalization and non-agglomeration of the Drug Unit.

In some embodiments, the Drug unit is not TZT-1027. In some embodiments,the Drug unit is not auristatin E, dolastatin 10, or auristatin PE.

Exemplary antibody-drug conjugate compounds have the followingstructures wherein “L” or “mAb-s-” represents an 158P1D7 MAb designatedHa15-10ac12 set forth herein:

or pharmaceutically acceptable salt thereof.

In some embodiments, the Drug Unit is a calicheamicin, camptothecin, amaytansinoid, or an anthracycline. In some embodiments the drug is ataxane, a topoisomerase inhibitor, a vinca alkaloid, or the like.

In some typical embodiments, suitable cytotoxic agents include, forexample, DNA minor groove binders (e.g., enediynes and lexitropsins, aCBI compound; see also U.S. Pat. No. 6,130,237), duocarmycins, taxanes(e.g., paclitaxel and docetaxel), puromycins, and vinca alkaloids. Othercytotoxic agents include, for example, CC-1065, SN-38, topotecan,morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin,echinomycin, combretastatin, netropsin, epothilone A and B,estramustine, cryptophysins, cemadotin, maytansinoids, discodermolide,eleutherobin, and mitoxantrone.

In some embodiments, the Drug is an anti-tubulin agent. Examples ofanti-tubulin agents include, auristatins, taxanes (e.g., Taxol®(paclitaxel), Taxotere® (docetaxel)), T67 (Tularik) and vinca alkyloids(e.g., vincristine, vinblastine, vindesine, and vinorelbine). Otherantitubulin agents include, for example, baccatin derivatives, taxaneanalogs (e.g., epothilone A and B), nocodazole, colchicine and colcimid,estramustine, cryptophycins, cemadotin, maytansinoids, combretastatins,discodermolide, and cleutherobin.

In certain embodiments, the cytotoxic agent is a maytansinoid, anothergroup of anti-tubulin agents. For example, in specific embodiments, themaytansinoid is maytansine or DM-1 (ImmunoGen, Inc.; see also Chari etal., 1992, Cancer Res. 52:127-131).

In certain embodiments, the cytotoxic or cytostatic agent is adolastatin. In certain embodiments, the cytotoxic or cytostatic agent isof the auristatin class. Thus, in a specific embodiment, the cytotoxicor cytostatic agent is MMAE (Formula XI). In another specificembodiment, the cytotoxic or cytostatic agent is AFP (Formula XVI).

In certain embodiments, the cytotoxic or cytostatic agent is a compoundof formulas XII-XXI or pharmaceutically acceptable salt thereof:

X.) Drug Loading

Drug loading is represented by p and is the average number of Drugmoieties per antibody in a molecule. Drug loading may range from 1 to 20drug moieties (D) per antibody. ADCs of the invention includecollections of antibodies conjugated with a range of drug moieties, from1 to 20. The average number of drug moieties per antibody inpreparations of ADC from conjugation reactions may be characterized byconventional means such as mass spectroscopy and, ELISA assay. Thequantitative distribution of ADC in terms of p may also be determined.In some instances, separation, purification, and characterization ofhomogeneous ADC where p is a certain value from ADC with other drugloadings may be achieved by means such as electrophoresis.

For some antibody-drug conjugates, p may be limited by the number ofattachment sites on the antibody. For example, where the attachment is acysteine thiol, as in the exemplary embodiments above, an antibody mayhave only one or several cysteine thiol groups, or may have only one orseveral sufficiently reactive thiol groups through which a linker may beattached. In certain embodiments, higher drug loading, e.g. p>5, maycause aggregation, insolubility, toxicity, or loss of cellularpermeability of certain antibody-drug conjugates. In certainembodiments, the drug loading for an ADC of the invention ranges from 1to about 8; from about 2 to about 6; from about 3 to about 5; from about3 to about 4; from about 3.1 to about 3.9; from about 3.2 to about 3.8;from about 3.2 to about 3.7; from about 3.2 to about 3.6; from about 3.3to about 3.8; or from about 3.3 to about 3.7. Indeed, it has been shownthat for certain ADCs, the optimal ratio of drug moieties per antibodymay be less than 8, and may be about 2 to about 5. See US 2005-0238649A1 (herein incorporated by reference in its entirety).

In certain embodiments, fewer than the theoretical maximum of drugmoieties are conjugated to an antibody during a conjugation reaction. Anantibody may contain, for example, lysine residues that do not reactwith the drug-linker intermediate or linker reagent, as discussed below.Generally, antibodies do not contain many free and reactive cysteinethiol groups which may be linked to a drug moiety; indeed most cysteinethiol residues in antibodies exist as disulfide bridges. In certainembodiments, an antibody may be reduced with a reducing agent such asdithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partialor total reducing conditions, to generate reactive cysteine thiolgroups. In certain embodiments, an antibody is subjected to denaturingconditions to reveal reactive nucleophilic groups such as lysine orcysteine.

The loading (drug/antibody ratio) of an ADC may be controlled indifferent ways, e.g., by: (i) limiting the molar excess of drug-linkerintermediate or linker reagent relative to antibody, (ii) limiting theconjugation reaction time or temperature, (iii) partial or limitingreductive conditions for cysteine thiol modification, (iv) engineeringby recombinant techniques the amino acid sequence of the antibody suchthat the number and position of cysteine residues is modified forcontrol of the number and/or position of linker-drug attachments (suchas thioMab or thioFab prepared as disclosed herein and in WO2006/034488(herein incorporated by reference in its entirety)).

It is to be understood that where more than one nucleophilic groupreacts with a drug-linker intermediate or linker reagent followed bydrug moiety reagent, then the resulting product is a mixture of ADCcompounds with a distribution of one or more drug moieties attached toan antibody. The average number of drugs per antibody may be calculatedfrom the mixture by a dual ELISA antibody assay, which is specific forantibody and specific for the drug. Individual ADC molecules may beidentified in the mixture by mass spectroscopy and separated by HPLC,e.g. hydrophobic interaction chromatography (see, e.g., Hamblett, K. J.,et al. “Effect of drug loading on the pharmacology, pharmacokinetics,and toxicity of an anti-CD30 antibody-drug conjugate,” Abstract No. 624,American Association for Cancer Research, 2004 Annual Meeting, Mar.27-31, 2004, Proceedings of the AACR, Volume 45, March 2004; Alley, S.C., et al. “Controlling the location of drug attachment in antibody-drugconjugates,” Abstract No. 627, American Association for Cancer Research,2004 Annual Meeting, Mar. 27-31, 2004, Proceedings of the AACR, Volume45, March 2004). In certain embodiments, a homogeneous ADC with a singleloading value may be isolated from the conjugation mixture byelectrophoresis or chromatography.

XI.) Methods of Determining Cytotoxic Effect of ADCs

Methods of determining whether a Drug or Antibody-Drug conjugate exertsa cytostatic and/or cytotoxic effect on a cell are known. Generally, thecytotoxic or cytostatic activity of a Antibody Drug conjugate can bemeasured by: exposing mammalian cells expressing a target protein of theAntibody Drug conjugate in a cell culture medium; culturing the cellsfor a period from about 6 hours to about 5 days; and measuring cellviability. Cell-based in vitro assays can be used to measure viability(proliferation), cytotoxicity, and induction of apoptosis (caspaseactivation) of the Antibody Drug conjugate.

For determining whether a Antibody Drug conjugate exerts a cytostaticeffect, a thymidine incorporation assay may be used. For example, cancercells expressing a target antigen at a density of 5,000 cells/well of a96-well plated can be cultured for a 72-hour period and exposed to 0.5μCi of ³H-thymidine during the final 8 hours of the 72-hour period. Theincorporation of ³H-thymidine into cells of the culture is measured inthe presence and absence of the Antibody Drug conjugate.

For determining cytotoxicity, necrosis or apoptosis (programmed celldeath) can be measured. Necrosis is typically accompanied by increasedpermeability of the plasma membrane; swelling of the cell, and ruptureof the plasma membrane. Apoptosis is typically characterized by membraneblebbing, condensation of cytoplasm, and the activation of endogenousendonucleases. Determination of any of these effects on cancer cellsindicates that a Antibody Drug conjugate is useful in the treatment ofcancers.

Cell viability can be measured by determining in a cell the uptake of adye such as neutral red, trypan blue, or ALAMAR™ blue (see, e.g., Pageet al., 1993, Intl. J. Oncology 3:473-476). In such an assay, the cellsare incubated in media containing the dye, the cells are washed, and theremaining dye, reflecting cellular uptake of the dye, is measuredspectrophotometrically. The protein-binding dye sulforhodamine B (SRB)can also be used to measure cytotoxicity (Skehan et al., 1990, J. Natl.Cancer Inst. 82:1107-12).

Alternatively, a tetrazolium salt, such as MTT, is used in aquantitative colorimetric assay for mammalian cell survival andproliferation by detecting living, but not dead, cells (see, e.g.,Mosmann, 1983, J. Immunol. Methods 65:55-63).

Apoptosis can be quantitated by measuring, for example, DNAfragmentation. Commercial photometric methods for the quantitative invitro determination of DNA fragmentation are available. Examples of suchassays, including TUNEL (which detects incorporation of labelednucleotides in fragmented DNA) and ELISA-based assays, are described inBiochemica, 1999, no. 2, pp. 34-37 (Roche Molecular Biochemicals).

Apoptosis can also be determined by measuring morphological changes in acell. For example, as with necrosis, loss of plasma membrane integritycan be determined by measuring uptake of certain dyes (e.g., afluorescent dye such as, for example, acridine orange or ethidiumbromide). A method for measuring apoptotic cell number has beendescribed by Duke and Cohen, Current Protocols in Immunology (Coligan etal. eds., 1992, pp. 3.17.1-3.17.16). Cells also can be labeled with aDNA dye (e.g., acridine orange, ethidium bromide, or propidium iodide)and the cells observed for chromatin condensation and margination alongthe inner nuclear membrane. Other morphological changes that can bemeasured to determine apoptosis include, e.g., cytoplasmic condensation,increased membrane blebbing, and cellular shrinkage.

The presence of apoptotic cells can be measured in both the attached and“floating” compartments of the cultures. For example, both compartmentscan be collected by removing the supernatant, trypsinizing the attachedcells, combining the preparations following a centrifugation wash step(e.g., 10 minutes at 2000 rpm), and detecting apoptosis (e.g., bymeasuring DNA fragmentation). (See, e.g., Piazza et al., 1995, CancerResearch 55:3110-16).

In vivo, the effect of a 158P1D7 therapeutic composition can beevaluated in a suitable animal model. For example, xenogenic cancermodels can be used, wherein cancer explants or passaged xenografttissues are introduced into immune compromised animals, such as nude orSCID mice (Klein et al., 1997, Nature Medicine 3: 402-408). For example,PCT Patent Application WO98/16628 and U.S. Pat. No. 6,107,540 describevarious xenograft models of human prostate cancer capable ofrecapitulating the development of primary tumors, micrometastasis, andthe formation of osteoblastic metastases characteristic of late stagedisease. Efficacy can be predicted using assays that measure inhibitionof tumor formation, tumor regression or metastasis, and the like.

In vivo assays that evaluate the promotion of apoptosis are useful inevaluating therapeutic compositions. In one embodiment, xenografts fromtumor bearing mice treated with the therapeutic composition can beexamined for the presence of apoptotic foci and compared to untreatedcontrol xenograft-bearing mice. The extent to which apoptotic foci arefound in the tumors of the treated mice provides an indication of thetherapeutic efficacy of the composition.

The therapeutic compositions used in the practice of the foregoingmethods can be formulated into pharmaceutical compositions comprising acarrier suitable for the desired delivery method. Suitable carriersinclude any material that when combined with the therapeutic compositionretains the anti-tumor function of the therapeutic composition and isgenerally non-reactive with the patient's immune system. Examplesinclude, but are not limited to, any of a number of standardpharmaceutical carriers such as sterile phosphate buffered salinesolutions, bacteriostatic water, and the like (see, generally,Remington's Pharmaceutical Sciences 16th Edition, A. Osal., Ed., 1980).

Therapeutic formulations can be solubilized and administered via anyroute capable of delivering the therapeutic composition to the tumorsite. Potentially effective routes of administration include, but arenot limited to, intravenous, parenteral, intraperitoneal, intramuscular,intratumor, intradermal, intraorgan, orthotopic, and the like. Apreferred formulation for intravenous injection comprises thetherapeutic composition in a solution of preserved bacteriostatic water,sterile unpreserved water, and/or diluted in polyvinylchloride orpolyethylene bags containing 0.9% sterile Sodium Chloride for Injection,USP. Therapeutic protein preparations can be lyophilized and stored assterile powders, preferably under vacuum, and then reconstituted inbacteriostatic water (containing for example, benzyl alcoholpreservative) or in sterile water prior to injection.

Dosages and administration protocols for the treatment of cancers usingthe foregoing methods will vary with the method and the target cancer,and will generally depend on a number of other factors appreciated inthe art.

XII.) Treatment of Cancer(s) Expressing 158P1D7

The identification of 158P1D7 as a protein that is normally expressed ina restricted set of tissues, but which is also expressed in cancers suchas those listed in Table I, opens a number of therapeutic approaches tothe treatment of such cancers.

Of note, targeted antitumor therapies have been useful even when thetargeted protein is expressed on normal tissues, even vital normal organtissues. A vital organ is one that is necessary to sustain life, such asthe heart or colon. A non-vital organ is one that can be removedwhereupon the individual is still able to survive. Examples of non-vitalorgans are ovary, breast, and prostate.

Expression of a target protein in normal tissue, even vital normaltissue, does not defeat the utility of a targeting agent for the proteinas a therapeutic for certain tumors in which the protein is alsooverexpressed. For example, expression in vital organs is not in and ofitself detrimental. In addition, organs regarded as dispensable, such asthe prostate and ovary, can be removed without affecting mortality.Finally, some vital organs are not affected by normal organ expressionbecause of an immunoprivilege. Immunoprivileged organs are organs thatare protected from blood by a blood-organ barrier and thus are notaccessible to immunotherapy. Examples of immunoprivileged organs are thebrain and testis.

Accordingly, therapeutic approaches that inhibit the activity of a158P1D7 protein are useful for patients suffering from a cancer thatexpresses 158P1D7. These therapeutic approaches generally fall intothree classes. The first class modulates 158P1D7 function as it relatesto tumor cell growth leading to inhibition or retardation of tumor cellgrowth or inducing its killing. The second class comprises variousmethods for inhibiting the binding or association of a 158P1D7 proteinwith its binding partner or with other proteins. The third classcomprises a variety of methods for inhibiting the transcription of a158P1D7 gene or translation of 158P1D7 mRNA.

Accordingly, Cancer patients can be evaluated for the presence and levelof 158P1D7 expression, preferably using immunohistochemical assessmentsof tumor tissue, quantitative 158P1D7 imaging, or other techniques thatreliably indicate the presence and degree of 158P1D7 expression.Immunohistochemical analysis of tumor biopsies or surgical specimens ispreferred for this purpose. Methods for immunohistochemical analysis oftumor tissues are well known in the art.

XIII.) 158P1D7 as a Target for Antibody-Based Therapy

158P1D7 is an attractive target for antibody-based therapeuticstrategies. A number of antibody strategies are known in the art fortargeting both extracellular and intracellular molecules (see, e.g.,complement and ADCC mediated killing as well as the use of intrabodies).Because 158P1D7 is expressed by cancer cells of various lineagesrelative to corresponding normal cells, systemic administration of158P1D7-immunoreactive compositions are prepared that exhibit excellentsensitivity without toxic, non-specific and/or non-target effects causedby binding of the immunoreactive composition to non-target organs andtissues. Antibodies specifically reactive with domains of 158P1D7 areuseful to treat 158P1D7-expressing cancers systemically, preferably asantibody drug conjugates (i.e. ADCs) wherein the conjugate is with atoxin or therapeutic agent.

Those skilled in the art understand that antibodies can be used tospecifically target and bind immunogenic molecules such as animmunogenic region of a 158P1D7 sequence shown in FIG. 1 . In addition,skilled artisans understand that it is routine to conjugate antibodiesto cytotoxic agents (see, e.g., Slevers et al. Blood 93:11 3678-3684(Jun. 1, 1999)). When cytotoxic and/or therapeutic agents are delivereddirectly to cells, such as by conjugating them to antibodies specificfor a molecule expressed by that cell (e.g. 158P1D7), the cytotoxicagent will exert its known biological effect (i.e. cytotoxicity) onthose cells.

A wide variety of compositions and methods for using antibody-cytotoxicagent conjugates to kill cells are known in the art. In the context ofcancers, typical methods entail administering to an mammal having atumor a biologically effective amount of a conjugate comprising aselected cytotoxic and/or therapeutic agent linked to a targeting agent(e.g. a 158P1D7 MAb, preferably Ha15-10ac12) that binds to an antigen(e.g. 158P1D7) expressed, accessible to binding or localized on the cellsurfaces. A typical embodiment is a method of delivering a cytotoxicand/or therapeutic agent to a cell expressing 158P1D7, comprisingconjugating the cytotoxic agent to an antibody that immunospecificallybinds to a 158P1D7 epitope, and, exposing the cell to the antibody drugconjugate (ADC). Another illustrative embodiment is a method of treatingan individual suspected of suffering from metastasized cancer,comprising a step of administering parenterally to said individual apharmaceutical composition comprising a therapeutically effective amountof an antibody conjugated to a cytotoxic and/or therapeutic agent.

Cancer immunotherapy using 158P1D7 antibodies can be done in accordancewith various approaches that have been successfully employed in thetreatment of other types of cancer, including but not limited to coloncancer (Arlen et al., 1998, Crit. Rev. Immunol. 18:133-138), multiplemyeloma (Ozaki et al., 1997, Blood 90:3179-3186, Tsunenari et al., 1997,Blood 90:2437-2444), gastric cancer (Kasprzyk et al., 1992, Cancer Res.52:2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J. Immunother.Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong et al., 1996, Leuk.Res. 20:581-589), colorectal cancer (Moun et al., 1994, Cancer Res.54:6160-6166; Velders et al., 1995, Cancer Res. 55:4398-4403), andbreast cancer (Shepard et al., 1991, J. Clin. Immunol. 11:117-127). Sometherapeutic approaches involve conjugation of naked antibody to a toxinor radioisotope, such as the conjugation of Y⁹1 or I¹³¹ to anti-CD20antibodies (e.g., Zevalin™, IDEC Pharmaceuticals Corp. or Bexxar™,Coulter Pharmaceuticals) respectively, while others involveco-administration of antibodies and other therapeutic agents, such asHerceptin™ (trastuzu MAb) with paclitaxel (Genentech, Inc.). In apreferred embodiment, the antibodies will be conjugated a cytotoxicagent, supra, preferably an aurastatin derivative designated MMAE(Seattle Genetics).

Although 158P1D7 antibody therapy is useful for all stages of cancer,antibody therapy can be particularly appropriate in advanced ormetastatic cancers. Treatment with the antibody therapy of the inventionis indicated for patients who have received one or more rounds ofchemotherapy. Alternatively, antibody therapy of the invention iscombined with a chemotherapeutic or radiation regimen for patients whohave not received chemotherapeutic treatment. Additionally, antibodytherapy can enable the use of reduced dosages of concomitantchemotherapy, particularly for patients who do not tolerate the toxicityof the chemotherapeutic agent very well. Fan et al. (Cancer Res.53:4637-4642, 1993), Prewett et al. (International J. of Onco.9:217-224, 1996), and Hancock et al. (Cancer Res. 51:4575-4580, 1991)describe the use of various antibodies together with chemotherapeuticagents.

Accordingly, preferred monoclonal antibodies used in the therapeuticmethods of the invention are those that are either fully human and thatbind specifically to the target 158P1D7 antigen with high affinity.Although the antibody drug conjugate of the invention is useful fortreating cancers in which the 158P1D7 is expressed, the antibody drugconjugate of the invention can be particulary therapeutically useful intreating bladder cancers.

XIV.) 158P1D7 ADC Cocktails

Therapeutic methods of the invention contemplate the administration ofsingle 158P1D7 ADCs as well as combinations, or cocktails, of differentMAbs (i.e. 158P1D7 MAbs or Mabs that bind another protein). Such MAbcocktails can have certain advantages inasmuch as they contain MAbs thattarget different epitopes, exploit different effector mechanisms orcombine directly cytotoxic MAbs with MAbs that rely on immune effectorfunctionality. Such MAbs in combination can exhibit synergistictherapeutic effects. In addition, 158P1D7 MAbs can be administeredconcomitantly with other therapeutic modalities, including but notlimited to various chemotherapeutic and biologic agents,androgen-blockers, immune modulators (e.g., IL-2, GM-CSF), surgery orradiation. In a preferred embodiment, the 158P1D7 MAbs are administeredin conjugated form.

158P1D7 ADC formulations are administered via any route capable ofdelivering the antibodies to a tumor cell. Routes of administrationinclude, but are not limited to, intravenous, intraperitoneal,intramuscular, intratumor, intradermal, and the like. Treatmentgenerally involves repeated administration of the 158P1D7 ADCpreparation, via an acceptable route of administration such asintravenous injection (IV), typically at a dose in the range, includingbut not limited to, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 mg/kg body weight. In general,doses in the range of 10-1000 mg MAb per week are effective and welltolerated.

Based on clinical experience with the Herceptin® (Trastuzumab) in thetreatment of metastatic breast cancer, an initial loading dose ofapproximately 4 mg/kg patient body weight IV, followed by weekly dosesof about 2 mg/kg IV of the MAb preparation represents an acceptabledosing regimen. Preferably, the initial loading dose is administered asa 90-minute or longer infusion. The periodic maintenance dose isadministered as a 30 minute or longer infusion, provided the initialdose was well tolerated. As appreciated by those of skill in the art,various factors can influence the ideal dose regimen in a particularcase. Such factors include, for example, the binding affinity and halflife of the MAbs used, the degree of 158P1D7 expression in the patient,the extent of circulating shed 158P1D7 antigen, the desired steady-stateantibody concentration level, frequency of treatment, and the influenceof chemotherapeutic or other agents used in combination with thetreatment method of the invention, as well as the health status of aparticular patient.

Optionally, patients should be evaluated for the levels of 158P1D7 in agiven sample (e.g. the levels of circulating 158P1D7 antigen and/or158P1D7 expressing cells) in order to assist in the determination of themost effective dosing regimen, etc. Such evaluations are also used formonitoring purposes throughout therapy, and are useful to gaugetherapeutic success in combination with the evaluation of otherparameters (for example, urine cytology and/or ImmunoCyt levels inbladder cancer therapy, or by analogy, serum PSA levels in prostatecancer therapy).

An object of the present invention is to provide 158P1D7 ADCs, whichinhibit or retard the growth of tumor cells expressing 158P1D7. Afurther object of this invention is to provide methods to inhibitangiogenesis and other biological functions and thereby reduce tumorgrowth in mammals, preferably humans, using such 158P1D7 ADCs, and inparticular using such 158P1D7 ADCs combined with other drugs orimmunologically active treatments.

XV.) Combination Therapy

In one embodiment, there is synergy when tumors, including human tumors,are treated with 158P1D7 ADCs in conjunction with chemotherapeuticagents or radiation or combinations thereof. In other words, theinhibition of tumor growth by a 158P1D7 ADC is enhanced more thanexpected when combined with chemotherapeutic agents or radiation orcombinations thereof. Synergy may be shown, for example, by greaterinhibition of tumor growth with combined treatment than would beexpected from a treatment of only 158P1D7 ADC or the additive effect oftreatment with a 158P1D7 ADC and a chemotherapeutic agent or radiation.Preferably, synergy is demonstrated by remission of the cancer whereremission is not expected from treatment either from a 158P1D7 ADC orwith treatment using an additive combination of a 158P1D7 ADC and achemotherapeutic agent or radiation.

The method for inhibiting growth of tumor cells using a 158P1D7 ADC anda combination of chemotherapy or radiation or both comprisesadministering the 158P1D7 ADC before, during, or after commencingchemotherapy or radiation therapy, as well as any combination thereof(i.e. before and during, before and after, during and after, or before,during, and after commencing the chemotherapy and/or radiation therapy).For example, the 158P1D7 ADC is typically administered between 1 and 60days, preferably between 3 and 40 days, more preferably between 5 and 12days before commencing radiation therapy and/or chemotherapy. However,depending on the treatment protocol and the specific patient needs, themethod is performed in a manner that will provide the most efficacioustreatment and ultimately prolong the life of the patient.

The administration of chemotherapeutic agents can be accomplished in avariety of ways including systemically by the parenteral and enteralroutes. In one embodiment, the 158P1D7 ADCs and the chemotherapeuticagent are administered as separate molecules. Particular examples ofchemotherapeutic agents or chemotherapy include cisplatin, dacarbazine(DTIC), dactinomycin, mechlorethamine (nitrogen mustard), streptozocin,cyclophosphamide, carmustine (BCNU), lomustine (CCNU), doxorubicin(adriamycin), daunorubicin, procarbazine, mitomycin, cytarabine,etoposide, methotrexate, 5-fluorouracil, vinblastine, vincristine,bleomycin, paclitaxel (taxol), docetaxel (taxotere), aldesleukin,asparaginase, busulfan, carboplatin, cladribine, dacarbazine,floxuridine, fludarabine, hydroxyurea, ifosfamide, interferon alpha,leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane,pegaspargase, pentostatin, pipobroman, plicamycin, streptozocin,tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracilmustard, vinorelbine, gemcitabine, chlorambucil, taxol and combinationsthereof.

The source of radiation, used in combination with a 158P1D7 ADC, can beeither external or internal to the patient being treated. When thesource is external to the patient, the therapy is known as external beamradiation therapy (EBRT). When the source of radiation is internal tothe patient, the treatment is called brachytherapy (BT).

The above described therapeutic regimens may be further combined withadditional cancer treating agents and/or regimes, for example additionalchemotherapy, cancer vaccines, signal transduction inhibitors, agentsuseful in treating abnormal cell growth or cancer, antibodies (e.g.Anti-CTLA-4 antibodies as described in WO/2005/092380 (Pfizer)) or otherligands that inhibit tumor growth by binding to IGF-1R, and cytokines.

When the mammal is subjected to additional chemotherapy,chemotherapeutic agents described above may be used. Additionally,growth factor inhibitors, biological response modifiers, anti-hormonaltherapy, selective estrogen receptor modulators (SERMs), angiogenesisinhibitors, and anti-androgens may be used. For example, anti-hormones,for example anti-estrogens such as Nolvadex (tamoxifen) or,anti-androgens such as Casodex(4′-cyano-3-(4-fluorophenylsulphonyl)-2-hydroxy-2-methyl-3-′-(trifluoromethyl)propionanilide)may be used.

The above therapeutic approaches can be combined with any one of a widevariety of surgical, chemotherapy or radiation therapy regimens. Thetherapeutic approaches of the invention can enable the use of reduceddosages of chemotherapy (or other therapies) and/or less frequentadministration, an advantage for all patients and particularly for thosethat do not tolerate the toxicity of the chemotherapeutic agent well.

XVI.) Kits/Articles of Manufacture

For use in the laboratory, prognostic, prophylactic, diagnostic andtherapeutic applications described herein, kits are within the scope ofthe invention. Such kits can comprise a carrier, package, or containerthat is compartmentalized to receive one or more containers such asvials, tubes, and the like, each of the container(s) comprising one ofthe separate elements to be used in the method, along with a label orinsert comprising instructions for use, such as a use described herein.For example, the container(s) can comprise an antibody that is or can bedetectably labeled. Kits can comprise a container comprising a DrugUnit. The kit can include all or part of the amino acid sequences inFIG. 2 , or FIG. 3 or analogs thereof, or a nucleic acid molecule thatencodes such amino acid sequences.

The kit of the invention will typically comprise the container describedabove and one or more other containers associated therewith thatcomprise materials desirable from a commercial and user standpoint,including buffers, diluents, filters, needles, syringes; carrier,package, container, vial and/or tube labels listing contents and/orinstructions for use, and package inserts with instructions for use.

A label can be present on or with the container to indicate that thecomposition is used for a specific therapy or non-therapeuticapplication, such as a prognostic, prophylactic, diagnostic orlaboratory application, and can also indicate directions for either invivo or in vitro use, such as those described herein. Directions and orother information can also be included on an insert(s) or label(s) whichis included with or on the kit. The label can be on or associated withthe container. A label a can be on a container when letters, numbers orother characters forming the label are molded or etched into thecontainer itself; a label can be associated with a container when it ispresent within a receptacle or carrier that also holds the container,e.g., as a package insert. The label can indicate that the compositionis used for diagnosing, treating, prophylaxing or prognosing acondition, such as a cancer of a tissue set forth in Table I.

The terms “kit” and “article of manufacture” can be used as synonyms.

In another embodiment of the invention, an article(s) of manufacturecontaining compositions, such as antibody(s), or antibody drugconjugates (ADCs) e.g., materials useful for the diagnosis, prognosis,prophylaxis and/or treatment of cancers of tissues such as those setforth in Table I is provided. The article of manufacture typicallycomprises at least one container and at least one label. Suitablecontainers include, for example, bottles, vials, syringes, and testtubes. The containers can be formed from a variety of materials such asglass, metal or plastic. The container can hold amino acid sequence(s),small molecule(s), nucleic acid sequence(s), cell population(s) and/orantibody(s). In another embodiment a container comprises an antibody,binding fragment thereof or specific binding protein for use inevaluating protein expression of 158P1D7 in cells and tissues, or forrelevant laboratory, prognostic, diagnostic, prophylactic andtherapeutic purposes; indications and/or directions for such uses can beincluded on or with such container, as can reagents and othercompositions or tools used for these purposes.

The container can alternatively hold a composition that is effective fortreating, diagnosis, prognosing or prophylaxing a condition and can havea sterile access port (for example the container can be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). The active agents in the composition can be anantibody capable of specifically binding 158P1D7 or an antibody drugconjugate specifically binding to 158P1D7.

The article of manufacture can further comprise a second containercomprising a pharmaceutically-acceptable buffer, such asphosphate-buffered saline, Ringer's solution and/or dextrose solution.It can further include other materials desirable from a commercial anduser standpoint, including other buffers, diluents, filters, stirrers,needles, syringes, and/or package inserts with indications and/orinstructions for use.

EXAMPLES

Various aspects of the invention are further described and illustratedby way of the several examples that follow, none of which is intended tolimit the scope of the invention.

Example 1 The 158P1D7 Antigen

The 158P1D7 gene sequence was discovered using Suppression SubtractiveHybridization (SSH) methods known in the art. The 158P1D7 SSH sequenceof 223 bp was identified from a bladder cancer pool minus normal bladdercDNA substraction. A full length cDNA clone for 158P1D7 was isolatedfrom a bladder cancer tissue pool. The cDNA is 2,555 bp in length andencodes an 841 amino acid ORF (See, FIG. 1 ). The 158P1D7 gene showshomology to SLITRK6 gene. For further reference see, U.S. Pat. No.6,863,892 (Agensys, Inc., Santa Monica, CA), U.S. Pat. No. 7,358,353(Agensys, Inc., Santa Monica, CA) and EP 1,311,675 (Agensys, Inc., SantaMonica, CA). For exemplary embodiments of the 158P1D7 antigen, see FIG.1 .

Example 2 Generation of 158P1 D7 Monoclonal Antibodies (MAbs)

In one embodiment, therapeutic Monoclonal Antibodies (“MAbs”) to 158P1D7and 158P1D7 variants comprise those that react with epitopes specificfor each protein or specific to sequences in common between the variantsthat would bind, internalize, disrupt or modulate the biologicalfunction of 158P1D7 or 158P1D7 variants, for example, those that woulddisrupt the interaction with ligands, substrates, and binding partners.Immunogens for generation of such MAbs include those designed to encodeor contain the extracellular domains or the entire 158P1D7 proteinsequence, regions predicted to contain functional motifs, and regions ofthe 158P1D7 protein variants predicted to be antigenic from computeranalysis of the amino acid sequence. Immunogens include peptides andrecombinant proteins such as tag5-158P1D7, a purified mammalian cellderived His tagged protein. In addition, cells engineered to expresshigh levels of 158P1D7, such as UMUC-158P1D7 or 3T3-158P1D7, are used toimmunize mice.

MAbs to 158P1D7 were generated using XenoMouse Technology® (AmgemFremont) wherein the murine heavy and kappa light chain loci have beeninactivated and a majority of the human heavy and kappa light chainimmunoglobulin loci have been inserted. The MAb designated Ha15-10ac12was generated from immunization of human y² producing XenoMice withrecombinant 3T3 cells expressing 158P1D7.

The 158P1D7 MAb Ha15-10ac12 specifically binds to 158P1D7 expressingcells (recombinant and endogenous) as well as recombinant 158P1D7protein by ELISA.

DNA coding sequences for 158P1D7 MAb Ha15-10ac12 was determined afterisolating mRNA from the respective hybridoma cells with Trizol reagent(Life Technologies, Gibco BRL).

Anti-158P1D7 Ha15-10ac12 heavy and light chain variable nucleic acidsequences were sequenced from the hybridoma cells using the followingprotocol. Ha15-10ac12 secreting hybridoma cells were lysed with Trizolreagent (Life Technologies, Gibco BRL). Total RNA was purified andquantified. First strand cDNAs was generated from total RNA with oligo(dT)₁₂-18 priming using the Gibco-BRL Superscript Preamplificationsystem. First strand cDNA was amplified using human immunoglobulinvariable heavy chain primers, and human immunoglobulin variable lightchain primers. PCR products were sequenced and the variable heavy andlight chain regions determined.

The nucleic acid and amino acid sequences of the variable heavy andlight chain regions are listed in FIG. 2 and FIG. 3 . Alignment ofHa15-10ac12 MAb to human Ig germline is set forth in FIG. 4A-4B.

Example 3 Expression of Ha15-10Ac12 Using Recombinant DNA Methods

To express Ha15-10ac12 MAb recombinantly in transfected cells,Ha15-10ac12 MAb variable heavy and light chain sequences were clonedupstream of the human heavy chain IgG2 and human light chain Igκconstant regions respectively. The complete Ha15-10ac12 MAb human heavychain and light chain cassettes were cloned downstream of the CMVpromoter/enhancer in a cloning vector. A polyadenylation site wasincluded downstream of the MAb coding sequence. The recombinantHa15-10ac12 MAb expressing constructs were transfected into CHO-K1SVcells. The Ha15-10ac12 related MAb secreted from recombinant CHO cellswas evaluated for binding to cell surface 158P1D7 by flow cytometry.UMUC-control and UMUC-158P1D7 cells were stained with Ha15-10ac12 MAbfrom either hybridoma or from CHO cells transfected with Ha15-10ac12heavy and light chain vector constructs. Binding was detected by flowcytometry.

Results show that the recombinantly expressed Ha15-10ac12 expressed inCHO cells binds 158P1D7 similarly to the Ha15-10ac12 purified fromhybridoma. The Ha15-10ac12 MAb secreted from recombinant cells was alsoevaluated for binding to 158P1D7 recombinant protein by ELISA. Bindingof Ha15-10ac12 to 158P1D7 protein was identical between MAb materialderived from CHO and from hybridoma cells.

The Chinese Hamster Ovary (CHO) cell producing an antibody designatedHa15-10ac12 was sent (via Federal Express) to the American Type CultureCollection (ATCC), P.O. Box 1549, Manassas, VA 20108 on 25 Jul. 2012 andassigned Accession number PTA-13102.

Example 4 Antibody Drug Conjugation of Ha15-10Ac12 MAb

The Ha15-10ac12 Mab (FIG. 2 ) was conjugated to an auristatin derivativedesignated MMAE (Formula XI) using a vc (Val-Cit) linker describedherein to create the antibody drug conjugate (ADC) of the inventiondesignated Ha15-10ac12vcMMAE using the following protocols. Theconjugation of the ve (Val-Cit) linker to the MMAE (Seattle Genetics,Seattle, WA) was completed using the general method set forth in TableIV to create the cytotoxic vcMMAE (see, US/2006/0074008).

Next, the antibody drug conjugate (ADC) of the invention designatedHa15-10ac12vcMMAE was made using the following protocols.

Briefly, the Ha15-10ac12 MAb in formulation buffer (10 mM acetate pH 5.0with 5% sorbitol) is buffer-exchanged to reduction buffer (25 mM sodiumborate, 300 mM sodium chloride, pH of 9.0±0.1). The Ha15-10ac12 MAb isthen partially reduced by adding 5 mM EDTA and 2.65 molar equivalents ofTCEP (relative to moles of Ha15-10ac12 MAb). This mixture is thenstirred at 37° C. for three (3) hours. Following disulfide reduction,the mixture is cooled to a target temperature of 15-17° C. and five (5)drug equivalents of vcMMAE per mole are added as a 4% (v/v) solution ofDMSO. After 60 to 75 minutes, the excess unreacted vc is quenched byadding N-Acetyl-L-Cysteine in the amount of 1 mole per mole of vcMMAEadded at the beginning of conjugation. After 15 minutes, theHa15-10ac12vcMMAE is adjusted to a target pH of 6.0-6.4 using aconcentrated histidine pH 5.2 buffer stock and filtered through a0.5/0.2 μm PES membrane to remove aggregated antibody-drug-conjugate.Immediately following filtration, tangential flow filtration isperformed to remove DMSO and quenched drug linker and to exchange theantibody-drug-conjugate to the buffer, 20 mM histidine (pH 6.0±0.1)containing 5.5% trehalose dehydrate. After tangential flow filtration,the Ha15-10ac12vcMMAE is diluted to a final concentration of 6±1 mg/mLand polysorbate 20 is added to a final concentration of 0.01%.

The resulting antibody drug conjugate (ADC) is designatedHa15-10ac12vcMMAE and has the following formula:

wherein MAb is Ha15-10ac12 (FIG. 2 and FIG. 3 ) and p is from 1 to 12.The preferred p value of the antibody drug conjugate set forth in thisExample is between 3.5 and 3.7.

Example 5 Characterization of Ha15-10ac12vcMMAE

Antibody Drug Conjugates that bind 158P1D7 were generated using theprocedures set forth in the example entitled “Antibody Drug Conjugationof Ha15-10ac12 MAb” and were screened, identified, and characterizedusing a combination of assays known in the art.

A. Affinity Determination by FACS

Ha15-10ac12vcMMAE was tested for its binding affinity to 158P1D7expressed on the surface of SW780 cells. Briefly, eleven (11) dilutionsof Ha15-10ac12vcMMAE are incubated with SW780 cells (50,000 cells perwell) overnight at 4° C. at a final concentration of 6.67 nM to 0.0001nM. At the end of the incubation, cells are washed and incubated withanti-hIgG-PE detection antibody for 45 min at 4° C. After washing theunbound detection antibodies, the cells are analyzed by FACS. MeanFlorescence Intensity (MFI) values were obtained as listed in (TableVI). MFI values were entered into Graphpad Prisim software and analyzedusing the one site binding (hyperbola) equation of Y=Bmax*X/(Kd+X) togenerate Ha15-10ac12vcMMAE saturation curve shown in (FIG. 13 ). Bmax isthe MFI value at maximal binding of Ha15-10ac12vcMMAE to 158P1D7; Kd isthe Ha15-10ac12vcMMAE binding affinity which is the concentration ofHa15-10ac12vcMMAE required to reach half-maximal binding.

The calculated affinity (Kd) of Ha15-10ac12vcMMAE to 158P1D7 expressedon the surface of SW780 cell is 0.005 nM.

B. FACS Binding

Ha15-10ac12vcMMAE was tested by FACS for its binding to 158P1D7expressed on the surface of UMUC, SW780 and CHP-212 cells. Briefly, thecells were harvested and plated at a concentration of 50,000 cells perwell. Ha15-10ac12vcMMAE was diluted to either 3 μg/mL (for UMUC andSW780 cells) or 10 μg/mL (for CHP-212 cells) and incubated with thecells (1 hour at 4° C.). At the end of the incubation, cells are washedand incubated with anti-hIgG-PE detection antibody for 1 hour at 4° C.After washing the unbound detection antibodies, the cells are analyzedby FACS. Mean Florescence Intensity (MFI) values were obtained (TableVII) and histograms are shown (FIG. 14 ).

Example 6 In Vitro Cell Cytotoxicity Mediated by Ha15-10ac12vcMMAE

The ability of Ha15-10ac12vcMMAE to mediate SLITRK-6 dependentcytotoxicity was evaluated using the human neuroblastoma CHP-212 cellline, which endogenously express SLITRK-6 and the human ovarian cancercell line, IGROV-1, which does not express SLITRK-6.

Briefly, the CHP-212 and IGROV-1 cells were seeded in 50 μl of completemedia, at a density of 1000 cell/well, onto 96 well plates and placed ina tissue culture incubator at 37 degrees C.; 5% CO₂. The next day a 2×stock solution of Ha15-10ac12vcMMAE and isotype control antibodyconjugated to vcMMAE were prepared in complete media and 50 μl of theserial dilutions of the ADCs were added to the appropriate wells. Thecells were treated with Ha15-10ac12vcMMAE and the isotype controlantibody conjugated to vcMMAE for six (6) days in a tissue cultureincubator at 37 degrees C.; 5% CO₂. At the end of the incubation period,12 l of Alamar Blue or Presto Blue was added to each well and incubatedfor four (4) hours. The plates were read using a BioTek Synergy H4 platereader using 540 Excitation and 590 Emission wavelengths.

These data show that the ADC entitled Ha15-10ac12vcMMAE can selectivelyinduce the cytotoxicity of the SLITRK-6 expressing CHP-212 cell linewhile it is unable to induce the cytotoxicity of the SLITRK-6non-expressing IGROV-1 cell line. Thus, these results indicate thatHa15-10ac12vcMMAE can selectively deliver a cytotoxic drug to 158P1D7expressing cells leading to their killing (FIG. 15 ).

In another experiment, human neuroblastoma cells, CHP-212, which expressSLITRK6 (target for Ha15-10ac12 MAb and M15-68(2) 18) (a.k.a. 68(18)1.1, See, WO 2004/072263) MAb), were incubated with the test articles,Ha15-10ac12vcMMAE and M15-68(2) 18, to demonstrate any in vitro cellkilling (cytotoxicity) activity of these articles. Briefly, ninety-six(96) well assay plates were seeded with 4000 CHP-212 cells/ml and testantibodies were added in a dilution series from 10,000 ng/ml down to0.006 ng/ml using a six (6) fold dilution over nine (9) points plus a0.0 ng/ml final test well. Wells were set up in triplicate. In addition,control antibodies and control cells (IGROV-1) were set up in a similarway for comparison with the test articles. The assay was allowed to runfor four (4) days before PrestoBlue (pigment that stains live cells) wasadded to the wells to give a colorimetric readout of cell viability.Percentage cell survival was then calculated in each well and data wasanalyzed using non-linear fitting of sigmoidal dose response. IC50values were calculated using Prism graphing software and cytotoxiceffects of Ha15-10ac12vcMMAE were compared with M15-68(2)₁₈ and othercontrols used in the experiment.

The results show that Ha15-10ac12vcMMAE posesses superior in vitrocytotoxicity when compared with the M15-68(2) 18 antibody and othercontrols. (FIG. 11 ). The IC50 for Ha15-10ac12vcMMAE was shown to be0.9076 and the IC50 for M15-68(2) 18 could not be calculated.

Example 7 Ha15-10ac12vcMMAE Inhibit Growth of Tumors In Vivo

The significant expression of 158P1D7 on the cell surface of tumortissues, together with its restrictive expression in normal tissuesmakes 158P1D7 a good target for antibody therapy and similarly, therapyvia ADC. Thus, the therapeutic efficacy of Ha15-10ac12vcMMAE in humanbladder, lung, breast, and glioblastoma cancer xenograft mouse models isevaluated.

Antibody drug conjugate efficacy on tumor growth and metastasisformation is studied in mouse cancer xenograft models (e.g. subcutaneousand orthotopically).

Subcutaneous (s.c.) tumors are generated by injection of 5×10⁴-10⁶cancer cells mixed at a 1:1 dilution with Matrigel (CollaborativeResearch) in the right flank of male SCID mice. To test ADC efficacy ontumor formation, i.e. ADC injections are started on the same day astumor-cell injections. As a control, mice are injected with eitherpurified human IgG or PBS; or a purified MAb that recognizes anirrelevant antigen not expressed in human cells. In preliminary studies,no difference is found between control IgG or PBS on tumor growth. Tumorsizes are determined by caliper measurements, and the tumor volume iscalculated as width²×Length/2, wherein width is the smallest dimensionand length is the largest dimension. Mice with subcutaneous tumorsgreater than 1.5 cm in diameter are sacrificed.

An advantage of xenograft cancer models is the ability to studyneovascularization and angiogenesis. Tumor growth is partly dependent onnew blood vessel development. Although the capillary system anddeveloping blood network is of host origin, the initiation andarchitecture of the neovasculature is regulated by the xenograft tumor(Davidoff et al., Clin Cancer Res. (2001) 7:2870; Solesvik et al., Eur JCancer Clin Oncol. (1984) 20:1295). The effect of antibody and smallmolecule on neovascularization is studied in accordance with proceduresknown in the art, such as by IHC analysis of tumor tissues and theirsurrounding microenvironment.

158P1D7 ADCs:

Monoclonal antibodies were raised against 158P1D7 as described in theExample entitled “Generation of 158P1D7 Monoclonal Antibodies (MAbs).”Further the MAbs are conjugated to a toxin as described in the Exampleentitled “Antibody Drug Conjugation of Ha15-10ac12 MAb” to formHa15-10ac12vcMMAE. The Ha15-10ac12vcMMAE is characterized by FACS, andother methods known in the art to determine its capacity to bind158P1D7.

Cell Lines and Xenografts:

The cells are maintained in DMEM, supplemented with L-glutamine and 10%FBS, as known in the art. The AG-B7, RT-4-XCL, and NCI-H322M-XCLxenografts are maintained by serial propagation in SCID mice. The SW780and RT-4-XCL are cell derived bladder cancer line(s) that were obtainedvia the A.T.C.C. (Manassas, VA). The AG-B7 and AG-B8 are patient derivedxenograft(s) derived from human bladder cancer specimens. One of skillin the art will appreciate that a plurality of experiments are performedin vivo on the ADCs of the invention. This is due, in part, that everyin vivo model, even if in the same disease indication, exhibits a levelof unpredictability to those of skill in the art. Thus, one of skill inthe art will appreciate that utilization of several types of in vivomodels will allow one of skill in the art to better assess the ADCs ofthe invention.

Evaluation of Ha15-10ac12vcMMAE in the Subcutaneous EstablishedXenograft Model of Human Bladder Cancer AG-B7 in SCID Mice

In this experiment, human bladder cancer AG-B7 cells (5×10⁶ cells permouse) were injected into the flanks of individual SCID mice and tumorswere allowed to grow untreated until they reached an approximate volumeof 250 mm³. At that point, animals were allocated to each group based ontumor volume at the time of treatment initiation to ensure similar meantumor size and variation in each group using Study Director Software(v.1.7; Studylog Systems, Inc., South San Francisco, CA). All ADCtreated groups received a single dose at 10 mg/kg by intravenous bolusinjection. Tumor growth in each group was monitored twice weekly usingcaliper measurements until study termination. Statistical analysis oftumor volumes was performed at the last time point when data from allgroups were available using a nonparametric analysis of variance (ANOVA)on the ranked data.

The results show that Ha15-10ac12vcMMAE demonstrated a potent inhibitoryeffect when compared to the non-treated control (p<0.0001) (FIG. 5 ).

Evaluation of Ha15-10ac12vcMMAE in the Subcutaneous Established HumanBladder Cancer RT-4-XCL Implanted in SCID Mice

In another experiment, human bladder cancer xenograft AG-B7 stock tumorswere harvested sterilely and minced in to small pieces (approximately 1mm³). Six pieces were implanted subcutaneously into the flanks ofindividual SCID mice. When the average tumor volumes reached apredetermined size of 100 mm³ in volume, animals were randomized intoADC treated groups and a non-treated control group (see tumor volumegraph) with similar mean tumor size and variation in each group usingStudy Director Software (v.1.7; Studylog Systems, Inc., South SanFrancisco, CA). All ADC treated groups, including two control ADCs,received a single dose at 5 mg/kg by intravenous bolus injection. Tumorgrowth in each group was monitored twice weekly using calipermeasurements until study termination. Statistical analysis of tumorvolumes was performed at the last time point when data from all groupswere available using a nonparametric analysis of variance (ANOVA) on theranked data.

The results show that Ha15-10ac12vcMMAE demonstrated a potent tumorinhibitory effect when compared to the non-treated control or to thecorresponding ADC control H3-1.4.1.2vcMMAE (both p<0.0001) (FIG. 6 ).

Evaluation of Ha15-10ac12vcMMAE in the Subcutaneous Established HumanLung Cancer NCI-H322M-XCL Implanted in SCID Mice

In another experiment, Human lung cancer NCI-H322M-XCL cells (2.5×10⁶cells per mouse) were injected into the flanks of individual SCID miceand tumors were allowed to grow untreated until they reached anapproximate volume of 200 mm³. At that point, animals were allocated toeach group based on tumor volume at the time of treatment initiation toensure similar mean tumor size and variation in each group using StudyDirector Software (v.1.7; Studylog Systems, Inc., South San Francisco,CA). All ADC groups including the two control ADCs were dosed at 3 mg/kgtwo times per week for a total of five doses by intravenous bolusinjection. Tumor growth in each group was monitored twice weekly usingcaliper measurements until study termination. Statistical analysis oftumor volumes was performed at the last time point when data from allgroups were available using a nonparametric analysis of variance (ANOVA)on the ranked data.

The results show that Ha15-10ac12vcMMAE demonstrated a potent inhibitoryeffect when compared to the vehicle control (p<0.0001) or to thecorresponding ADC control Ha3-12bc1.1vcMMAE (p=0.0041) (FIG. 7 ).

Efficacy of Ha15-10ac12vcMMAE in Subcutaneous Established XenograftModel of Human Bladder Cancer AG-B7 in SCID Mice

In another experiment, Human bladder cancer xenograft AG-B7 stock tumorswere harvested sterilely and minced into small pieces (approximately 1mm³). Five pieces were implanted subcutaneously into the flanks ofindividual SCID mice. Tumors were allowed to grow untreated until theyreached an approximate volume of 200 mm³. At that point, animals wereallocated to each group based on tumor volume at the time of treatmentinitiation to ensure similar mean tumor size and variation in each groupusing Study Director Software (v.1.7; Studylog Systems, Inc., South SanFrancisco CA). All ADC groups including the two control ADCs were dosedat 0.5 mg/kg twice a week for a total of seven doses by intravenousbolus injection. Tumor growth in each group was monitored twice weeklyusing caliper measurements until study termination. Statistical analysisof tumor volumes was performed at the last time point when data from allgroups were available using a nonparametric analysis of variance (ANOVA)on the ranked data.

The results show that Ha15-10ac12vcMMAE produced from hybridoma(Ha15-10ac12.1vcMMAE) and CHO cells (Ha15-10ac12vcMMAE) bothdemonstrated a potent tumor inhibitory effect when compared to eitherthe vehicle control (p<0.0001) or to the corresponding ADC controlHa3-12abc1vcMMAE (p<0.0001) (FIG. 8 ).

Efficacy of Ha15-10ac12vcMMAE in Subcutaneous Established XenograftModel of Human Bladder Cancer SW780 in SCID Mice

In another experiment, human bladder cancer xenograft SW780 cells wereimplanted into the flanks of SCID mice and tumors were allowed to growuntil they reached approximate volume of 200 mm³. At that point, animalswere allocated to the treatment groups according to tumor volume at thetime of treatment initiation to ensure similar mean tumor size andvariation in each group. Allocation of mice into treatment groups wasaided by Study Director Software (v.1.7; Studylog Systems, Inc., SouthSan Francisco CA) to help sixe match the mice. All ADC groups includingthe two control ADCs were dosed, by intravenous bolus injection, at 1mg/kg at the beginning of the study. Tumor growth in each group wasmonitored twice weekly using caliper measurements until studytermination. Statistical analysis of tumor volumes was performed at thelast time point when data from all groups were available using anonparametric analysis of variance (ANOVA) on the ranked data.

The results show that Ha15-10ac12vcMMAE has superior tumor inhibitoryactivity than the Ha15-10ac12 MAb. Further, it can be concluded thatHa15-10ac12 MAb has no tumor inhibitory effect as the growth dynamics ofthe tumor in this treatment group follows that of the isotype controlantibodies. Further, after a single dose of 1 mg/kg, Ha15-10ac12vcMMAEshowed statistically significant growth inhibition when compared to theisotype control ADC (p<0.001) (FIG. 10 ).

Efficacy of Ha15-10ac12vcMMAE in Subcutaneous Established XenograftModel of Patient Derived Human Bladder Cancer AG-B8 in SCID Mice

In another experiment, patient derived human bladder cancer AG-B8 tumorpieces were implanted into the flanks of SCID mice and tumors wereallowed to grow until they reached approximate volume of 200 mm³. Atthat point, animals were allocated to the treatment groups according totumor volume at the time of treatment initiation to ensure similar meantumor size and variation in each group. Allocation of mice intotreatment groups was aided by Study Director Software (v.1.7; StudylogSystems, Inc., South San Francisco CA) to help sixe match the mice. AllADC groups including the two control ADCs were dosed, by intravenousbolus injection, at 5 mg/kg at the beginning of the study (day zero(0)). Tumor growth in each group was monitored twice weekly usingcaliper measurements until study termination. Statistical analysis oftumor volumes was performed at the last time point when data from allgroups were available using a nonparametric analysis of variance (ANOVA)on the ranked data.

The results show that Ha15-10ac12vcMMAE has superior tumor inhibitoryactivity than either non-treated control(s) (p<0.0001) or to othercorresponding ADC gamma-2 control(s) (p<0.0001). Additionally,Ha15ac12vcMMAE was also showed superior statistically significant effectwhen compared to Ha15-10ac12mcMMAF (p<0.0458) (FIG. 12 ).

CONCLUSION

In summary, FIGS. 5-8, 10, and 12 , show that the 158P1D7 ADC entitledHa15-10ac12vcMMAE significantly inhibited the growth of tumors cellsthat express 158P1D7 when compared to control ADCs. Thus, theHa15-10ac12vcMMAE can be used for therapeutic purposes to treat andmanage cancers set forth in Table I Specifically, these results indicatethe Ha15-10ac12vcMMAE had inhibitory effect on various types of bladdercancer models, showing it can be particularly therapeutically useful inthe treatment of bladder cancer.

Example 8 Human Clinical Trials for the Treatment and Diagnosis of HumanCarcinomas Through Use of 158P1D7 ADCs

158P1D7 ADCs are used in accordance with the present invention whichspecifically bind to 158P1D7, and are used in the treatment of certaintumors, preferably those listed in Table I. In connection with each ofthese indications, two clinical approaches are successfully pursued.

I.) Adjunctive therapy: In adjunctive therapy, patients are treated with158P1D7 ADCs in combination with a chemotherapeutic or anti-neoplasticagent and/or radiation therapy or a combination thereof. Primary cancertargets, such as those listed in Table T, are treated under standardprotocols by the addition of 158P1D7 ADCs to standard first and secondline therapy. Protocol designs address effectiveness as assessed by thefollowing examples, including but not limited to, reduction in tumormass of primary or metastatic lesions, increased progression freesurvival, overall survival, improvement of patients health, diseasestabilization, as well as the ability to reduce usual doses of standardchemotherapy and other biologic agents. These dosage reductions allowadditional and/or prolonged therapy by reducing dose-related toxicity ofthe chemotherapeutic or biologic agent. 158P1D7 ADCs are utilized inseveral adjunctive clinical trials in combination with thechemotherapeutic or anti-neoplastic agents.

II.) Monotherapy: In connection with the use of the 158P1D7 ADCs inmonotherapy of tumors, the 158P1D7 ADCs are administered to patientswithout a chemotherapeutic or anti-neoplastic agent. In one embodiment,monotherapy is conducted clinically in end-stage cancer patients withextensive metastatic disease. Protocol designs address effectiveness asassessed by the following examples, including but not limited to,reduction in tumor mass of primary or metastatic lesions, increasedprogression free survival, overall survival, improvement of patientshealth, disease stabilization, as well as the ability to reduce usualdoses of standard chemotherapy and other biologic agents.

Dosage

Dosage regimens may be adjusted to provide the optimum desired response.For example, a single bolus may be administered, several divided dosesmay be administered over time or the dose may be proportionally reducedor increased as indicated by the exigencies of the therapeuticsituation. It is especially advantageous to formulate parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit containing a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the antibody and/or ADCand the particular therapeutic or prophylactic effect to be achieved,and (b) the limitations inherent in the art of compounding such anactive compound for the treatment of sensitivity in individuals.

An exemplary, non limiting range for a therapeutically effective amountof an 158P1D7 ADC administered in combination according to the inventionis about 0.5 to about 10 mg/kg, about 1 to about 5 mg/kg, at least 1mg/kg, at least 2 mg/kg, at least 3 mg/kg, or at least 4 mg/kg. Otherexemplary non-limiting ranges are for example about 0.5 to about 5mg/kg, or for example about 0.8 to about 5 mg/kg, or for example about 1to about 7.5 mg/kg. The high dose embodiment of the invention relates toa dosage of more than 10 mg/kg. It is to be noted that dosage values mayvary with the type and severity of the condition to be alleviated, andmay include single or multiple doses. It is to be further understoodthat for any particular subject, specific dosage regimens should beadjusted over time according to the individual need and the professionaljudgment of the person administering or supervising the administrationof the compositions, and that dosage ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed composition.

Clinical Development Plan (CDP)

The CDP follows and develops treatments of 158P1D7 ADCs in connectionwith adjunctive therapy or monotherapy. Trials initially demonstratesafety and thereafter confirm efficacy in repeat doses. Trials are openlabel comparing standard chemotherapy with standard therapy plus 158P1D7ADCs. As will be appreciated, one non-limiting criteria that can beutilized in connection with enrollment of patients is 158P1D7 expressionlevels in their tumors as determined by biopsy.

As with any protein or antibody infusion-based therapeutic, safetyconcerns are related primarily to (i) cytokine release syndrome, i.e.,hypotension, fever, shaking, chills; (ii) the development of animmunogenic response to the material (i.e., development of humanantibodies by the patient to the antibody therapeutic, or HAMAresponse); and, (iii) toxicity to normal cells that express 158P1D7.Standard tests and follow-up are utilized to monitor each of thesesafety concerns. 158P1D7 ADCs are found to be safe upon humanadministration.

Example 9 Detection of 158P1D7 Protein in Cancer Patient Specimens byIHC

Expression of 158P1D7 protein by immunohistochemistry was tested inpatient tumor specimens from (i) bladder, (ii) breast, (iii) lung, and(iv) glioblastoma cancer patients. Briefly, formalin fixed, paraffinwax-embedded tissues were cut into four (4) micron sections and mountedon glass slides. The sections were de-waxed, rehydrated and treated withcitra antigen retrieval solution (Biogenex, San Ramon, CA) in theEZ-Retriever microwave (Biogenex, San Ramon, CA) for 15 minutes at 95°C. Sections were then treated with 3% hydrogen peroxide solution toinactivate endogenous peroxidase activity. Serum-free protein block(Dako, Carpenteria, CA) was used to inhibit non-specific binding priorto incubation with monoclonal mouse anti-158P1D7 antibody or an isotypecontrol. Subsequently, the sections were treated with the SuperSensitive™ Polymer-horseradish peroxidase (HRP) Detection System whichconsists of an incubation in Super Enhancer™ reagent followed by anincubation with polymer-HRP secondary antibody conjugate (BioGenex, SanRamon, CA). The sections were then developed using the DAB kit(BioGenex, San Ramon, CA). Nuclei were stained using hematoxylin, andanalyzed by bright field microscopy. Specific staining was detected inpatient specimens using the 158P1D7 immunoreactive antibody, asindicated by the brown staining. (See, FIGS. 9(A), 9(C), 9(E), and 9(G).In contrast, the control antibody did not stain either patient specimen.(See, FIGS. 9(B), 9(D), 9(F), and 9(H).

The results show expression of 158P1D7 in the tumor cells of patientbladder, breast, lung, and glioblastoma cancer tissues. These resultsindicate that 158P1D7 is expressed in human cancers and that antibodiesdirected to this antigen and the antibody drug conjugate designatedHa15-10ac12vcMMAE are useful for diagnostic and therapeutic purposes.(FIG. 9 ).

Throughout this application, various website data content, publications,patent applications and patents are referenced. (Websites are referencedby their Uniform Resource Locator, or URL, addresses on the World WideWeb.) The disclosures of each of these references are herebyincorporated by reference herein in their entireties.

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any that are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

Tables

TABLE I Tissues that express 158P1D7 when malignant. Glioblastoma LungBladder Breast

TABLE II Amino Acid Abbreviations SINGLE LETTER THREE LETTER FULL NAME FPhe phenylalanine L Leu leucine S Ser serine Y Tyr tyrosine C Cyscysteine W Trp tryptophan P Pro proline H His histidine Q Gln glutamineR Arg arginine I Ile isoleucine M Met methionine T Thr threonine N Asnasparagine K Lys lysine V Val valine A Ala alanine D Asp aspartic acid EGlu glutamic acid G Gly glycine

TABLE III Amino Acid Substitution Matrix Adapted from the GCG Software9.0 BLOSUM62 amino acid substitution matrix (block substitution matrix).The higher the value, the more likely a substitution is found inrelated, natural proteins. A C D E F G H I K L M N P Q R S T V W Y . 4 0−2 −1 −2 0 −2 −1 −1 −1 −1 −2 −1 −1 −1 1 0 0 −3 −2 A 9 3 4 2 3 3 1 3 1 13 3 3 3 1 1 1 2 2 C 6 2 −3 −1 −1 −3 −1 −4 −3 1 −1 0 −2 0 −1 −3 −4 −3 D 5−3 −2 0 −3 1 −3 −2 0 −1 2 0 0 −1 −2 −3 −2 E 6 −3 −1 0 −3 0 0 −3 −4 −3 −3−2 −2 −1 1 3 F 6 −2 −4 −2 −4 −3 0 −2 −2 −2 0 −2 −3 −2 −3 G 8 3 1 3 2 1 20 0 1 2 3 2 2 H 4 −3 2 1 −3 −3 −3 −3 −2 −1 3 −3 −1 I 5 −2 −1 0 −1 1 2 0−1 −2 −3 −2 K 4 2 −3 −3 −2 −2 −2 −1 1 −2 −1 L 5 −2 −2 0 −1 −1 −1 1 −1 −1M 6 2 0 0 1 0 3 4 2 N 7 −1 −2 −1 −1 −2 −4 −3 P 5 1 0 −1 −2 −2 −1 Q 5 −1−1 −3 −3 −2 R 4 1 −2 −3 −2 S 5 0 −2 −2 T 4 −3 −1 V 11 2 W 7 Y

TABLE IV General Method for Synthesis of vcMMAE Where: AA1 = Amino Acid1 AA2 = Amino Acid 2 AA5 = Amino Acid 5 DIL = Dolaisoleuine DAP =Dolaproine Linker = Val-Cit (vc)

TABLE V Positions CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 asidentified by the Kabat, Chothia, and Contact schemes, respectively. ForCDR-H1, residue numbering is given listed using both the Kabat andChothia numbering schemes. CDR Kabat Chothia Contact CDR-L1 L24--L34L24--L34 L30--L36 CDR-L2 L50--L56 L50--L56 L46--L55 CDR-L3 L89--L97L89--L97 L89--L96 CDR-H1 H31--H35B H26--H32 . . . 34 H30--H35B (KabatNumbering¹) CDR-H1 H31--H35 H26--H32 H30--H35 (Chothia Numbering²)CDR-H2 H50--H65 H52--H56 H47--H58 CDR-H3 H95--H102 H95--H102 H93--H101

TABLE VI Mean Florescence Intensity (MFI) values in FACS assay. nMHa15-10ac12vcMMAE 6.667 155.0 2.222 149.6 0.741 140.9 0.247 133.8 0.082124.0 0.027 107.6 0.0091 87.0 0.0030 60.8 0.0010 25.7 0.0003 14.9 0.00014.3

TABLE VII Ha15-10ac12vcMMAE FACS MFA Values for Binding Assay UMUC-AGS15 SW780 CHP-212 MFI 115 159 142

1. (canceled)
 2. An antibody drug conjugate comprising an antibody orantigen binding fragment thereof which binds to a 158P1D7 protein,wherein the antibody or antigen binding fragment is conjugated tomonomethyl auristatin E (MMAE), and wherein the antibody or antigenbinding fragment thereof comprises: (i) a heavy chain variable regioncomprising complementarity determining regions (CDRs) consisting of theamino acid sequences of the CDRs in the heavy chain variable regionsequence set forth in SEQ ID NO:7 and a light chain variable regioncomprising an amino acid sequence at least 80% homologous to the lightchain variable region amino acid sequence set forth in SEQ ID NO:8; (ii)a heavy chain variable region comprising complementarity determiningregions (CDRs) consisting of the amino acid sequences of the CDRs in theheavy chain variable region sequence set forth in SEQ ID NO:7 and alight chain variable region consisting of the amino acid sequenceranging from position 1 to position 113 of SEQ ID NO:8; (iii) a heavychain variable region comprising complementarity determining regions(CDRs) consisting of the amino acid sequences of the CDRs in the heavychain variable region sequence set forth in SEQ ID NO:7 and a lightchain consisting of the amino acid sequence ranging from position 1 toposition 219 of SEQ ID NO:8; (iv) a heavy chain variable regioncomprising complementarity determining regions (CDRs) consisting of theamino acid sequences of the CDRs in the heavy chain variable regionsequence set forth in SEQ ID NO:7 and a light chain variable regionconsisting of the amino acid sequence of the light chain variable regionof an antibody produced by a Chinese Hamster Ovary (CHO) cell depositedunder ATCC Accession No. PTA-13102; (v) a heavy chain variable regioncomprising complementarity determining regions (CDRs) consisting of theamino acid sequences of the CDRs in the heavy chain variable regionsequence set forth in SEQ ID NO:7 and a light chain consisting of theamino acid sequence of a light chain of an antibody produced by aChinese Hamster Ovary (CHO) deposited under ATCC Accession No.PTA-13102; (vi) a heavy chain variable region comprising an amino acidsequence at least 80% homologous to the heavy chain variable regionamino acid sequence set forth in SEQ ID NO:7 and a light chain variableregion comprising CDRs consisting of the amino acid sequences of theCDRs in the light chain variable region sequence set forth in SEQ IDNO:8; (vii) a heavy chain variable region consisting of the amino acidsequence ranging from position 1 to position 120 of SEQ ID NO:7 and alight chain variable region comprising CDRs consisting of the amino acidsequences of the CDRs in the light chain variable region sequence setforth in SEQ ID NO:8; (viii) a heavy chain consisting of the amino acidsequence ranging from position 1 to position 446 of SEQ ID NO:7 and alight chain variable region comprising CDRs consisting of the amino acidsequences of the CDRs in the light chain variable region sequence setforth in SEQ ID NO:8; (ix) a heavy chain variable region consisting ofthe amino acid sequence of the heavy chain variable region of anantibody produced by a Chinese Hamster Ovary (CHO) cell deposited underAmerican Type Culture Collection (ATCC) Accession No. PTA-13102 and alight chain variable region comprising CDRs consisting of the amino acidsequences of the CDRs in the light chain variable region sequence setforth in SEQ ID NO:8; (x) a heavy chain consisting of the amino acidsequence of the heavy chain of an antibody produced by a Chinese HamsterOvary (CHO) cell deposited under ATCC Accession No. PTA-13102 and alight chain variable region comprising CDRs consisting of the amino acidsequences of the CDRs in the light chain variable region sequence setforth in SEQ ID NO:8; (xi) a heavy chain variable region comprisingcomplementarity determining regions (CDRs) consisting of the amino acidsequences of the CDRs set forth in SEQ ID NO:7 according to Kabatnumbering and a light chain variable region comprising an amino acidsequence at least 80% homologous to the light chain variable regionamino acid sequence set forth in SEQ ID NO:8; (xii) a heavy chainvariable region comprising complementarity determining regions (CDRs)consisting of the amino acid sequences of the CDRs set forth in SEQ IDNO:7 according to Kabat numbering and a light chain consisting of theamino acid sequence ranging from position 1 to position 219 of SEQ IDNO:8; (xiii) a heavy chain variable region comprising complementaritydetermining regions (CDRs) consisting of the amino acid sequences of theCDRs set forth in SEQ ID NO:7 according to Kabat numbering and a lightchain variable region consisting of the amino acid sequence of the lightchain variable region of an antibody produced by a Chinese Hamster Ovary(CHO) cell deposited under ATCC Accession No. PTA-13102; (xiv) a heavychain variable region comprising complementarity determining regions(CDRs) consisting of the amino acid sequences of the CDRs set forth inSEQ ID NO:7 according to Kabat numbering and a light chain consisting ofthe amino acid sequence of a light chain of an antibody produced by aChinese Hamster Ovary (CHO) deposited under ATCC Accession No.PTA-13102; (xv) a heavy chain variable region comprising an amino acidsequence at least 80% homologous to the heavy chain variable regionamino acid sequence set forth in SEQ ID NO:7 and a light chain variableregion comprising CDRs consisting of the amino acid sequences of theCDRs set forth in SEQ ID NO:8 according to Kabat numbering; (xvi) aheavy chain consisting of the amino acid sequence ranging from position1 to position 446 of SEQ ID NO:7 and a light chain variable regioncomprising CDRs consisting of the amino acid sequences of the CDRs setforth in SEQ ID NO:8 according to Kabat numbering; (xvii) a heavy chainvariable region consisting of the amino acid sequence of the heavy chainvariable region of an antibody produced by a Chinese Hamster Ovary (CHO)cell deposited under American Type Culture Collection (ATCC) AccessionNo. PTA-13102 and a light chain variable region comprising CDRsconsisting of the amino acid sequences of the CDRs set forth in SEQ IDNO:8 according to Kabat numbering; or (xviii) a heavy chain consistingof the amino acid sequence of the heavy chain of an antibody produced bya Chinese Hamster Ovary (CHO) cell deposited under ATCC Accession No.PTA-13102 and a light chain variable region comprising CDRs consistingof the amino acid sequences of the CDRs set forth in SEQ ID NO:8according to Kabat numbering.
 3. The antibody drug conjugate of claim 2,wherein the antigen binding fragment thereof is an Fab, F(ab′)2, Fv, orscFv fragment.
 4. The antibody drug conjugate of claim 2, wherein theantibody is a fully human antibody.
 5. The antibody drug conjugate ofclaim 2, wherein the antibody is recombinantly produced.
 6. The antibodydrug conjugate of claim 2, wherein the antibody or antibody bindingfragment thereof is conjugated to the MMAE via a linker.
 7. The antibodydrug conjugate of claim 6, wherein the linker comprises valinecitrulline.
 8. The antibody drug conjugate of claim 6, wherein theantibody drug conjugate has the following structure:

s wherein L-represents the antibody or antigen binding fragment thereofand p ranges from 1 to
 10. 9. The antibody drug conjugate of claim 8,wherein the p ranges from 2 to
 5. 10. A pharmaceutical composition thatcomprises a therapeutically effective amount of the antibody drugconjugate of claim 2 and a pharmaceutically acceptable excipient. 11.The pharmaceutical composition of claim 10, wherein the composition isfor treatment of glioblastoma cancer, lung cancer, bladder cancer, orbreast cancer.
 12. The pharmaceutical composition of claim 10, whereinthe composition is administered in combination with radiation or achemotherapeutic agent.
 13. The pharmaceutical composition of claim 10,further comprising a chemotherapeutic agent in a human unit dose form.14. A method of treating cancer in a subject, comprising administeringto said subject a therapeutically effective amount of the antibody drugconjugate of claim 2
 15. The method of claim 14, wherein the cancerexpresses 158P1D7.
 16. The method of claim 14, wherein the subject is ahuman subject.
 17. The method of claim 14, comprising administeringabout 1 to about 5 mg/kg of the antibody drug conjugate to the subject.18. The method of claim 14, wherein the cancer is selected from thegroup consisting of glioblastoma, lung cancer, bladder cancer, andbreast cancer.
 19. The method of claim 18, wherein the cancer is bladdercancer.